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Roles of metallothionein and zinc transporters in the homeostasis of histochemically reactive zinc in… Hsi, Sharon Wei-Yu 2004

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Roles of metallothionein and zinc transporters in the homeostasis of histochemically reactive zinc in mice by Sharon Wei-Yu Hsi B.Sc. (Dietetics), The University of British Columbia, 2002 A THESIS SUBMITTED IN PARTICAL FULFILMENT OF T H E RERQUIREMENTS . FOR THE DEGREE OF MASTER OF SCIENCE in THE F A C U L T Y OF G R A D U A T E STUDIES (Human Nutrition) T H E UNIVERSITY OF BRITISH COLUMBIA December, 2004 © Sharon Wei-Yu Hsi, 2004 A B S T R A C T Zinc is an essential trace mineral required for the normal function of many zinc-containing binding proteins. There are two intracellular pools of zinc: the non-exchangeable pool of zinc and the labile intracellular pool of zinc (LIPZ). In animal tissues, LIPZ is known as histochemically reactive zinc. The abundance of LIPZ has been shown to be critical to cellular functions such as cell proliferation and apoptosis. However, the mechanisms involved in LIPZ regulation are not clear. Since both metallothionein (MT) and, to some degree, zinc transporters have been shown to play important roles in zinc homeostasis, we hypothesized that the abundance of histochemically reactive zinc in mice will be homeostatically regulated through the interplay between M T and zinc transporters. The objective was to investigate the role of M T and zinc transporters in the homeostatic regulation of histochemically reactive zinc^ in response to various zinc intakes in mice. Twenty-three-day old metallothionein knockout (MT-KO) and the control metallothionein-wild type (MT-WT) mice were fed low- (2 mg Zn/kg diet), adequate- (30 mg Zn/kg diet), or high-zinc (150 mg Zn/kg diet) diet. MT-WT mice were maintained on their assigned diet for 2, 4, 7, or 21 days, and M T - K O mice were maintained on their assigned diet for 4 days before the liver and small intestine were removed for analyses. In M T - W T mice, body weight gain and feed intake were unaffected by zinc intake, but bone zinc concentration was reduced at low-zinc intake and increased at high-zinc intake. High-zinc intake caused zinc toxicity in M T - K O mice. In MT-WT mice, M T concentrations reflected dietary zinc intake in both the liver and small intestine. The abundance of histochemically reactive zinc in small intestine, but not that in the liver, also reflected zinc intake. The abundance of small intestinal histochemically reactive zinc was higher in MT-WT mice than that in M T - K O ii mice at each dietary zinc intake. Zinc transporters mRNA levels were generally not affected by dietary zinc intakes in both M T - K O and MT-WT mice. Overall, the data suggested that M T , but not zinc transporters, plays a role in the homeostatic regulation of histochemically reactive zinc in mice. iii T A B L E O F C O N T E N T S Abstract ii Table of Contents .....iv List of Tables '. vii List of Figures viii Acknowledgements x L INTRODUCTION 1 1. Literature Review 3 1.1. Zinc 3 1.1.1. Physiological functions of zinc 3 1.1.2. Distribution of body zinc in humans 7 1.1.3. Intracellular zinc 7 a) Labile intracellular zinc, 8 b) Assessment of labile intracellular zinc 9 c) Size of labile intracellular zinc 13 1.2. Metallothionein 15 1.2.1. MTisoforms 16 1.2.2. Basal M T concentration 16 1.2.3. Induction of M T 17 1.2.4. Intracellular location of M T 19 1.2.5. Functions of M T 20 1.3. Zinc homeostasis 21 1.3.1. Absorption and excretion 22 iv 1.3.2. Zinc and M T 23 a) Dietary zinc intake and M T 24 b) M T adaptation to dietary zinc intake 25 1.3.3. Zinc transporters 27 a) Zinc exporters 28 b) Zinc importers 31 c) Effects of zinc intake on zinc transporters 32 1.4. Dietary zinc, M T , and LIPZ 34 2. Hypothesis 35 3. Objective 37 H . R O L E O F M E T A L L O T H I O N E I N I N T H E H O M E O S T A S I S O F H I S T O C H E M I C A L L Y R E A C T I V E Z I N C I N M I C E 38 1. Introduction 38 2. Materials and Methods 41 3. Results 51 4. Discussion 67 H I . F U T U R E D I R E C T I O N S 106 1. Metallothionein is part of the histochemically reactive zinc 106 2. MT-3 and MT-4 proteins might have played an important role in the regulation of zinc homeostasis in M T - K O mice 107 3. M T - K O mice might have used Zincosomes as a strategy to control zinc toxicity.... 107 R E F E R E N C E S 109 A P P E N D I C E S 124 v Appendix! Dietary composition 124 Appendix II. Metallothionein concentration in M T - K O mice 129 vi LIST O F T A B L E S Table II-1. Effect of dietary zinc intake on body weight gain and feed intake in MT-WT and M T - K O mice 82 Table AI-1. Dietary Composition 125 Table AI-2. Composition of mineral premix without zinc 126 Table AI-3. Composition of vitamin premix 127 Table AI-4. Composition of zinc premix 128 Table AI-5. Composition of biotin premix 128 Table AII-1. Metallothionein concentration in M T - K O mice 130 vii LIST O F FIGURES Figure 1-1. Zinc transporters 28 Figure 1-2. Rationale 36 Figure II-1. Effect of dietary zinc intake and treatment duration on bone zinc concentration 83 Figure II-2. Effect of dietary zinc intake and treatment duration on metallothionein level in the liver 84 Figure II-3. Effect of dietary zinc intake and treatment duration on metallothionein level in small intestine 85 Figure II-4. Zinquin-dependent fluorescence of the liver in MT-WT mice 86 Figure II-5. Zinquin-dependent fluorescence of small intestine in MT-WT mice 88 Figure II-6. RT-PCR analysis on the effect of dietary zinc intake and treatment duration on ZnT-1 mRNA level in the liver 90 Figure II-7. RT-PCR analysis on the effect of dietary zinc intake and treatment duration on ZnT-1 mRNA level in small intestine 92 Figure II-8. RT-PCR analysis on the effect of dietary zinc intake and treatment duration on ZnT-2 mRNA level in the liver 94 Figure II-9. RT-PCR analysis on the effect of dietary zinc intake and treatment duration on ZnT-2 mRNA level in small intestine 96 Figure 11-10. RT-PCR analysis on the effect of dietary zinc intake and treatment duration on ZnT-4 mRNA level in the liver 98 Figure 11-11. RT-PCR analysis on the effect of dietary zinc intake and treatment duration on ZnT-4 mRNA level in small intestine 100 viii Figure 11-12. RT-PCR analysis on the effect of dietary zinc intake and treatment duration on Nramp2 mRNA level in the liver 102 Figure 11-13. RT-PCR analysis on the effect of dietary zinc intake and treatment duration on Nramp2 mRNA level in small intestine 104 ix A C K N O W L E D G E M E N T S I wish to thank Dr. Zhaoming Xu for his encouragement, support, patience, and guidance. Thank you for teaching me how to develop critical thinking skills and the proper ways to solve problems. Moreover, thank you for letting me realize my weaknesses and giving me a chance to correct them. I wish to specially thank Dr. Eunice Li-Chan and Dr. Kathy Keiver for their time and their constructive comments. I also wish to thank Dr. Ryna Levy-Milne for reviewing my thesis and offering her comments. Moreover, I wish to thank Dr. Kim Cheng for chairing my defense and offering his advice on statistical analyses. I wish to deeply thank my parents and my brother for their love and support. Thank you for believing in me. I also wish to thank Beryl Pai, Caesar Chow, and Luke Chu for their help. I wish to especially thank Betty Hu for her help. This project may not have been carried out as smoothly as it did without the help from Betty. CHAPTER I I N T R O D U C T I O N Zinc, an essential trace mineral to humans and other animals, is present in all body fluids and tissues. At cellular level, zinc is mainly bound to macromolecules such as proteins. Some of the bindings between zinc and proteins are tight, while others are loose. Metallothionein (MT) is the most well studied and characterized zinc-binding protein. M T has been proposed to function as a zinc storage protein and play an important role in zinc homeostasis. Besides binding to proteins, a small portion of cellular zinc is in its free ionic form (Zn ), although the exact quantity of free zinc ions is not known. Loosely-bound zinc and ionic zinc are collectively referred to as the labile intracellular pool of zinc (LIPZ). In animal tissues, LEPZ is referred to as histochemically reactive zinc. Being an essential trace mineral, zinc is important to many metabolic activities. For example, zinc is required for cellular proliferation and structural integrity (Cousins et al., 2003; Klaus and Lothar, 2003). Thus, zinc deficiency leads to the occurrence of many clinical symptoms in humans and animals. One of the most profound symptoms of zinc deficiency in humans and animals is growth retardation, but the mechanisms involved are not known. Our lab has shown that in 3T3 cells, zinc deficiency results in low abundance of LIPZ and impaired DNA synthesis and cell proliferation, while zinc supplementation results in an increase in LEPZ abundance along with an increase in DNA synthesis and cell proliferation (Paski & Xu, 2001). These observations suggest that the abundance of LEPZ can be influenced by zinc nutrition and the abundance of LEPZ is associated with and critical to cell proliferation. 1 Although zinc deficiency is known to result in growth retardation and the abundance of LIPZ appears to be critical to cell proliferation, it is not known how zinc nutrition influences the abundance of L P Z and how LIPZ is homeostatically regulated in response to various zinc intakes. Since M T is known to play a role in zinc homeostasis, this project investigates the role of M T in the homeostasis of histochemically reactive zinc in mice. 2 1. Literature Review 1.1 Zinc (Zn) Zinc, a trace mineral, is present in all organs, tissues, and body fluids in animals and humans (Cousins, 1997). Dietary Reference Intake (DRI) for zinc is 14 mg/day for men and 9 mg/day for women. The absorption of dietary zinc is influenced by various factors, including dietary zinc intake levels, protein intake levels, the presence of inhibitory factors (e.g. phytate and fiber) and divalent cations (e.g. iron, calcium, copper, and cadmium), and gastric emptying and intestinal transit time (Coyle et al., 1999; Lonnerdal, 2000). It is estimated that the total zinc content in human adult body is approximately 1.5 to 2.5 g. About 99 % of the body zinc is present intracellularly (Vallee 1993; Cousins, 1997). Zinc metabolism is influenced by a variety of factors, such as stress, inflammation, infection, etc. (Pattison and Cousins, 1986). For example, in murine aortic endothelial cells activated with proinflammatory cytokines, nitric oxide is produced. The cytotoxic nitric oxide leads to a nuclear translocation of M T and a further release of labile zinc in the nucleus (Spahl et al., 2003). Moreover, mice injected with bacterial endotoxin lipopolysaccharide (LPS) have an increase in intestinal zinc retention and a reduction in zinc excretion compared to mice without LPS injection (Philcox et al., 2000). 1.1.1 Physiological functions of zinc Zinc exerts very diverse and seemingly unrelated physiological functions. Dietary zinc deficiency in humans results in many clinical symptoms, including anorexia, growth retardation, skin lesions, mental disorders, depressed immune functions, etc. (Hambidge et al., 1986; Vallee and Falchuk, 1993; Dufner-Beattie et al., 2003). Zinc deprivation renders 3 organisms more susceptible to injury induced by a variety of oxidative stress (Powell, 2000; Tapiero and Tew, 2003). Human fibroblasts cells (IMR90) grown in zinc-deficient medium prepared by removing zinc from regular Fetal Bovine Serum with Chelex-100 show an oxidative DNA damage (e.g. glutathione peroxidase up-regulation) and a down-regulation of several mitochondrial electron transport chain proteins (e.g. ATP synthase; Ho et al., 2003). Rats fed a zinc-deficient diet (4 mg Zn/kg diet) for 14 days have an increased sensitivity to copper-mediated lipoprotein oxidation (DiSilverstro and Blostein-Fujh, 1997). Supplementation of zinc to otherwise deficient diets can prevent and reverse these symptoms. For example, zinc supplementation at 20 mg/day prevents growth faltering in children (Roy et al., 1997). Infants age 6-9 months who receive 10 mg Zn/day for an average of 6.9 months have a 0.75 cm greater length increment than those who receive placebo (Rivera et al., 1998). Furthermore, Sazawal et al. (2001) also found that a daily zinc supplementation of 5 mg as zinc sulfate for low birth weight infants from 30 to 284 days of age significantly reduces mortality rate. Zinc exerts these seemingly diverse physiological functions probably because zinc is a component of numerous zinc metalloenzymes. Zinc is a component of over 300 metalloenzymes (Vallee and Falchuk, 1993). These zinc metalloenzymes can be found in each of the six major categories of enzymes designated by the International Union of Biochemistry and Molecular Biology Commission on Enzyme Nomenclature (Hambidge et al., 1986). However, more studies are needed to demonstrate the relationship between zinc deficiency and the functionality of these zinc metalloenzymes. In these zinc metalloenzymes, zinc plays catalytic, structural, or regulatory roles (Cousins, 1997). Zinc exerts its catalytic role in various zinc metalloenzymes, e.g. R N A nucleotide transferases and carbonic anhydrases (DiSilversto et al., 1983). In this role, a zinc 4 ion is located at the active site of an enzyme, where it directly participates in the catalysis of enzymatic reactions (Vallee and Falchuk, 1993; McCall et al., 2000). The removal of zinc inactivates these zinc metalloenzymes, while supplementation of zinc restores the activities of these zinc metalloenzymes. For example, leucine aminopeptidase (bovine lens) is a zinc metalloenzyme containing two zinc atoms per subunit. Removal of these zinc atoms by dialysis inactivates the enzyme; however, re-addition of zinc to the medium fully restores the activity of the enzyme (Carpenter and Vahl, 1973). Similarly, the removal of zinc from phosphomannose isomerase using E D T A inhibits the activity of the enzyme, while the addition of EDTA-inhibitor reverses the inhibition (Gracy and Noutmann, 1968). In some metalloenzymes, e.g. Cu/Zn superoxide dismutase (Cu/Zn SOD), zinc is required for the structural stability of the enzyme. This function of zinc is known as the structural role of zinc (Vallee and Falchuk, 1993). Cu/Zn SOD catalyzes the dismutation of superoxide radicals, which is constantly formed during aerobic metabolism (McCord and Fridovich, 1969). In alcohol dehydrogenase, there are four zinc atoms in each enzyme molecule. Two of the zinc atoms exert the catalytic role, while the other two stabilize the structure (Groff and Gropper, 1999). Besides stabilizing metalloenzymes, zinc is also involved in maintaining the structure of a large number of transcription factors, known as zinc-finger proteins. In these proteins, zinc binds to the cysteine and/or histidine residues of the protein, forming finger-like structures. This finger-like structure facilitates the binding of these transcription factors to D N A double helix and, ultimately, gene transcription (Klug and Schwabe, 1995). The zinc-finger motif can mediate RNA binding and protein to protein interactions (Ladomery and Dellarie, 2002). Removal of zinc from zinc-finger proteins may result in a loss of function 5 for these transcription factors and DNA binding (Zeng et al., 1991; Cousins, 1997). These zinc-finger proteins are one of the largest classes of DNA-binding proteins involved in the control of gene expression (Zeng et al., 1991). At least 3% of the ~ 32,000 identified genes in the human genome encode proteins with zinc-finger domains (Maret, 2003). The multifunctional zinc-finger proteins are often linked to development and diseases. WT1 is an activator and repressor of transcription. It directly induces amphiregulin, which encodes a growth and differentiating factor of the epidermal growth factor (EFG) family (Lee et al., 1999). On the other hand, it inhibits the expression of cyclin E , a crucial enzyme that activates cyclin-dependent kinase 2 in cell cycle (Loeb et al., 2002). WT1 zinc-finger mutations are implicated in Wilm's tumor and Frasier Syndrome (Ladomery and Dellarie, 2003). ZNF74, which has a C 2 - H 2 zinc-finger domain, plays a role in RNA processing and maturation (Grondin et al., 1996). In mouse, Zfp265, which contains a (DW)C4 zinc-finger, is involved in the regulation of renin mRNA processing and stability in response to physiological changes (Ladomery and Dellarie, 2003). (DW)C4 zinc-finger motif is highly expressed in the brain and central nervous system during embryogenesis (Stolow and Haynes, 1995). Transcription factor Yin Yang 1 (YY1) contains multiple zinc-fingers. These zinc-fingers are necessary for D N A binding (Bushmeyer et al., 1995). Another example is CTCF, which is involved in, gene activation and repression. A mutation in the zinc-finger domains in C T C F has been found in Wilm's tumors, in which the DNA-binding specificity is altered (Filippova et al., 2002). Moreover, estrogen and glucocorticoid receptors also contain zinc-finger motifs (Klug and Schwabe, 1995). Since zinc is a critical component in zinc-finger proteins, the effect of zinc concentration (zinc intake or zinc concentration in culture medium) on zinc-finger proteins 6 has also been studied. For example, the T lymphocytes signal transduction pathway contains several zinc-finger proteins, such as protein kinase C and p56 l c k (Pernelle et al., 1991; Csermelly et al., 1998). A zinc-deficient diet (< 1 mg Zn/kg diet) is found to significantly elevate the expression of T lymphocyte p56 l c k in female mice (Lepage et al., 1999). The DNA binding domains of both peroxisome proliferators-activated receptors (PPAR) and its binding partner retinoid X receptor (RXR) contain two zinc-fingers (Meerarani et al., 2003). Zinc chelation by N, N, N ' , N'-tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN) decreases PPARy expression, while zinc supplementation reverses the expression of PPARy (Meerarani et al., 2003). Zinc has also been proposed to regulate the expression of certain genes, such as M T (Cousins, 1997). However, little is known about this regulator role of zinc. The only well-studied example is its role in the induction of the expression of M T or MT-like proteins. 1.1.2 Distribution of body zinc in humans In humans, about 85% of the whole body zinc is found in bone and muscle (Miller et al., 1994; King et al., 2000). The skin and liver zinc together contributes about 11% of the total body zinc (King et al., 2000). The remaining 2 - 4% of the body zinc is present in other tissues (e.g. kidneys ~ 0.7%, brain ~ 1.5% and hair ~ 0.1%; Miller et al., 1994). 1.1.3 Intracellular zinc The cellular distribution of zinc is not firmly established. It is estimated that approximately 30 - 40% of the cellular zinc is localized in the nucleus, 50% of the cellular zinc is in the cytosol and cytosolic organelles, and the remaining 10 - 20% is associated with 7 membranes (Beyersmann and Haase, 2001). Cellular zinc can also be categorized based on its dynamics: the essentially non-exchangeable zinc and the labile zinc. The majority (~ 90%) of the intracellular zinc is found in the non-exchangeable pool of zinc, which consists of zinc that is tightly bound within the tertiary protein structure of zinc metalloenzymes (Miller et al., 1994; Truong-Tran et al., 2000; Truong-Tran et al., 2003) and zinc-finger proteins (Klug and Schwabe, 1995). On the other hand, labile intracellular pool of zinc (LEPZ) contains zinc that is more readily exchangeable. a) Labile intracellular zinc. LEPZ consists of free ionic zinc and zinc that is loosely bound to macromolecules. LEPZ constitutes approximately 5 - 15% of the total cellular zinc depending on the tissue, as LEPZ size varies with tissues or cell types (Budde et al., 1997). Zinc from LIPZ is dynamically involved in many zinc-dependent processes, such as signal transduction and neurotransmission (Truong-Tran et al., 2003). The actual size and the homeostatic mechanisms of LIPZ are presently not known, although the concentration of intracellular free ionic zinc is estimated to be in picomolar or nanomolar range (Maret, 2000; Malaiyandi et al., 2004). Some evidence suggests that some of the intracellular zinc ions are "stored" in subcellular vesicles, which are referred to as "zincosomes" (Truong-Tran et al., 2000; Beyersmann and Haase, 2001). Intense vesicular fluorescent staining using Zinquin [(2-methyl-8-/»-toluenesulphonamido-6-quinolyloxy) acetic acid] in C6 rat glioma cells indicates a high amount of labile zinc in zincosomes (Haase and Beyersmann, 1999; Haase and Beyersmann, 2002). However, zincosomes are not well defined experimentally and their characteristics are essentially unknown thus far. For example, it is not known how zincosomes are formed, whether zincosomes are membrane-bound, what their physiological 8 significance is, how zinc is transported into and out of the structure, and how zincosomes interact with other intracellular organelles. b) Assessment of labile intracellular zinc. Evidence has shown that zinc is not only a structural component of many metalloenzymes, but the intracellular availability of zinc controls the activities of many enzymes within the cell (Maret et al., 1999). Assessing the concentration of intracellular free zinc as well as understanding the mechanisms for regulating intracellular zinc homeostasis have recently become possible due to the recent advances in the development of various fluorescence probes. In biological systems, there is an abundance of Mg2"1" (~2 mM) and C a 2 + (~ 1 uM to 3 mM) that can interfere with Z n 2 + determination. Other metal ions, such as C u 2 + , C d 2 + , and H g 2 + also have the potential of interfering; however, they are typically present at lower concentrations than zinc (Thompson et al., 2002). The criteria for good probes include: 1) metal selectivity or specificity; 2) probe stability; 3) probe binding aflfinity; and 4) ease of delivery to the target and cell permeability. The probes employed and developed so far can be categorized into 4 groups: 1) divalent metal ion fluorescent probes, such as Fura-2, Quin-3, and Mag-indo-1; 2) photoinduced electron transfer-based fluorescent (or aminofluorescein-based) probes, such as Newport Green, ZnAF-1, Zinpry 1 and 2, and ZnACF-1; 3) peptide-, protein-, and chelator/fluorophore- enhanced fluorescent probes (Walkup et al., 2000; Kimura and Aoki, 2001); and 4) toluenesulfonamide quinoline fluorescent probes, such as TSQ, Zinquin, and TFLZn. No single probe developed to date is perfect for examining intracellular free zinc, as each type of probe has its strengths and limitations. 9 Divalent metal ion fluorimetric fluorescent probes are used to examine intracellular divalent cations. These probes effectively detect C a 2 + (e.g. Indo-1) and M g 2 + (e.g. Mag-fura-2; Grynkiewiez et al., 1985). Owing to a tighter binding affinity for zinc, these probes can also detect zinc. Thompson et al. (2002) found that the Kd of Fura-2 for zinc binding is approximately 0.1 nM, while that for calcium binding is 80 nM. These probes are also ratiometric indicators, and intracellular free zinc can therefore be quantified by relating the intensities at two different excitation or emission wavelengths to the analyte concentration (Thompson et al., 2002). However, since these probes are sensitive to both zinc and other divalent metal ions, and the concentrations of other metals, such as C a 2 + and M g 2 + , are higher than zinc, the possibility that other metal ions can interfere with zinc determination cannot be ruled out. Furthermore, the binding affinity of the probe and the intracellular concentration of metal ions need to be considered when using these probes to detect free zinc. It is difficult to completely exclude the confounding interference from changes in other metal ions. For example, it is possible for M g 2 + to confound the result when Mag-fura-5 is used to detect free zinc, as the intracellular M g 2 + level in resting neuron cells (0.5 - 1 mM) is close to the Kd of Mag-fura-5 for M g 2 + (2.6 mM; Brocard et al., 1993; Sensi et al., 1997). Although new zinc probes modified from these divalent metal ions probes have been developed (e.g. FuraZin-1), and they do not form complexes with C a 2 + or M g 2 + at millimolar range, the effect of C a 2 + to Z n 2 + concentration ratio on the result still needs to be investigated. The photoinduced electron transfer-based fluorescent probes perturb an electron transfer-based quenching process upon binding of the analyte to zinc (Huston et al., 1988). These probes are more zinc specific, as they use the coordination of metals with nitrogen rather than carboxylic acid group as the basis of metal binding. Since carboxylic acid 10 residues on divalent cation dyes interact with divalent cations and the interaction has proven to be difficult to separate, nitrogen-based dyes are not as C a 2 + or M g 2 + specific (Reynolds, 2004). However, these probes can still detect other metals. For example, Newport Green can bind to Fe 2 + , C u 2 + , and C d 2 + (Reynolds, 2004), and its affinity for zinc is low (Kd approximately 1 uM; Canzoniero et al., 1997). Zinpry-1 is zinc sensitive, cell-permeable, and can be passively loaded into cells (Walkup et al., 2000; Burdette et al., 2001). Its Kd for zinc is estimated to be ~ 1 nM (Walkup et al., 2000). Zinpry-1 is suitable for staining live cells, as the bright fluorescence of Zinpry-1 can be excited at visible wavelengths (Hirano, et al., 2000). However, Zinpry-1 preferably stains the Golgi or a Golgi associated vesicle, and fluorescence can be induced by protonation within vesicular membranes, which gives high background brightness (Burdette et al., 2001). Peptide-enhanced zinc-fluorophore can be synthesized by covalently attaching a peptide containing a zinc-finger motif with a dansylamide residue (approximate Kd at 1.4 x 10"10 M at pH 7; Kimura and Aoki, 2001). The presence of M g 2 + and C o 2 + did not interfere with zinc analysis; however, the zinc-finger peptide is vulnerable to air oxidation and redox active metals (e.g. copper; Kimura and Aoki, 2001). The fluorescent image of free Z n 2 + can also be achieved using an indicator system consisting of apo-carbonic anhydrase (apoCAII) and a fluorescent inhibitor of the enzyme, A B D - N (Thompson et al., 2000). In the absence of Zn , a weak reddish fluorescence typical of A B D - N alone is exhibited. However, upon the binding of zinc to apoCAII {Kd = 4 pM), which then promotes the binding of A B D - N to apoCAII (Kd =0.9 uM), a significant blue shift in excitation and emission is observed (Chen and Kernohan, 1967; Thompson et al., 2000). Although apoCAII is very selective for Z n 2 + , Hg 2 + and C u 2 + bind more tightly to apoCAII than Zn 2 + . In addition, apoCAII can only be 11 used to measure the release of zinc from the cell because apoCAII is not cell-permeable (Thompson et al., 2000). Quinoline sulfonamide fluorescent probes are widely used to examine intracellular free zinc. Fluorescence is conferred on quinoline sulfonamide by zinc chelation by the two nitrogens. The probes remain essentially nonfluorescent in the absence of Z n 2 + , and the binding of zinc allows the free ligand of the analyte to exhibit fluorescence, and the intensity increases upon greater zinc binding (Fahrni and O'Halloran, 1999; Snitsarev et al., 2001). TSQ [6-methoxy-(8-/7-toluenesulfonamido)-quinoline] was first used as a histochemical stain for the mossy fibers of the hippocampus and heart tissue sections (Frederickson et al , 1987). Zinquin [(2-methyl-8-/?-toluenesulphonamido-6-quinolyloxy) acetic acid] is a derivative of TSQ with an extra methyl group and ester to improve cellular retention (Zalewski et al., 1994). The Kd of Zinquin for zinc is estimated to be ~ 1 uM (Zalewski et al., 1993). Although Zinquin is capable of detecting free Z n 2 + at around 4 pM, it is suggested that Zinquin is most ideal as a fluorophore for probing free Z n 2 + concentration between 100 pM and 10 n M (Fahrni and O'Halloran, 1999). Zinquin binds not only to free Z n 2 + , but also to loosely bound zinc, such as MT-bound zinc (Walkup et al., 2000). However, the amount of MT-bound zinc chelatable by Zinquin is currently unknown. Zinquin cannot cross the nuclear membrane to detect nuclear free Z n 2 + (Haase and Beyersmann, 2002). Moreover, although C d 2 + concentration is low in normal conditions, C d 2 + has been reported to enhance fluorescence when C d 2 + is added into the growing medium (Zalewski et al., 1993; Brand and Kleineke, 1996). TFLZn is synthesized as a water-soluble analogue of TSQ. The fluorescence emission increased ~ 100-fold in rat hippocampal slices in the presence of zinc (100 p.M), while the fluorescence was unchanged in the presence of Mg 2 + ' C d 2 + , Co 2 + , C u 2 + , 12 or Fe 3 + (Budde et al:, 1997). However, TFLZn [iV-(6-methoxy-8-quinolyl)-p-carboxybenzoylsulphonamide] has a lower zinc binding affinity compared to Zinquin (Kd ~ 20 uM), which makes it less likely for TFLZn to bind to loosely-bound zinc (Frederickson, 1989). Furthermore, TFLZn was found to have poor loading capacity in cultured cortical neurons and poor cellular retention (Canzoniero et al., 1997). Quinoline-based probes are able to permeate freely across membranes (Snitsarev et al., 2001). However, common limitations for all quinoline-based probes are: 1) the abundance of intracellular free zinc cannot be quantified; and 2) these probes are not ideal for staining live cells, as their near-ultraviolet excitation (~ 350 nm) can damage live cells (Burdette et al., 2001). As reviewed above, various types of fluorescent probes are available for assessing intracellular labile zinc. However, each probe has its own strengths and limitations. The choice of adequate probe therefore should be carefully evaluated based on experimental conditions so that an appropriate probe can help reach the objectives of the study with minimal limitations. c) Concentration of labile intracellular zinc. The concentration of labile intracellular Z n 2 + has been estimated using a variety of probes reviewed above. However, the values vary due to different types of cells examined, different probes used, and different experimental designs. For example, using Fura-2, the intracellular free Z n 2 + concentration in rat hepatocytes is estimated at 1.26 ± 0.27 uM (Brand and Kleineke, 1996). Using 6 5 Z n 2 + flux, basal free Zn in erythrocytes is estimated at 1.5 to 32 pM (Simons, 1991). The amount of "chelatable" free Z n 2 + depends on the binding affinity and cell-permeability of the probe. For example, using Zinquin spectrofluorimetry, labile zinc in rat thymocytes and human 13 chronic lymphocytic leukemia cells are estimated at 14.4 ± 7.8 pmol/106 cells and 20.4 ± 8 . 1 pmol/106 cells, respectively (Zalewski et al., 1993). Upon electrical stimulation, which increases the probe's cell permeability, free Z n 2 + i n Fura-2-stained GH3 pituitary tumor cells increases from 0.4 to 2 nM/3 x 106 cells (Atar et al., 1995). LIPZ concentration is also influenced by metabolic activities. When 3T3 cells are stimulated with growth factors platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and insulin-line growth factor-1 (IGF-1) to proliferate, LIPZ concentration increases along with an increased DNA synthesis and cell proliferation (Paski and Xu, 2002). Changes in LIPZ concentration have also been linked to apoptosis, the programmed cell death. Various studies have shown an increase in apoptosis when intracellular zinc is deficient. Intracellular zinc depletion caused by membrane-permeable zinc chelator TPEN activates caspase-1 and causes apoptosis in retinal neurons and astrocytes (Hyun et al., 2000). Peritoneal mast cells (RPMC) treated with TPEN to deplete labile zinc are more susceptible to apoptotic inducer (e.g. butyrate) than those that are not treated with TPEN (Ho et al., 2004). Zinquin fluorescence further confirms the removal of labile zinc by TPEN (Ho et al., 2004). While intracellular zinc deficiency is associated with increased apoptosis, zinc supplementation protects cells from apoptotic death. For example, in the hippocampal neurons in gerbils with transient global ischemia, zinc administration of 20 mg ZnC^/kg body weight significantly reduces the nuclear damage and subsequent neuronal death (Matsushita et al., 1996). In the presence of zinc specific ionophores, pyrithione and DIQ (5,7-diiodo-8-hydroxyquinoline), apoptosis is suppressed by as little as 5-25 uM Z n 2 + (without zinc ionophores, no suppression was observed at Z n 2 + less than 500 uM) (Zalewski et al., 1991). Since zinc tightly bound to proteins is not readily available for exchange, these 14 observations suggest that LIPZ is more readily available to support metabolic events such as cell proliferation and apoptosis. Although increasing evidence suggests that LIPZ is critical in participating in cellular metabolic activities, our knowledge in LIPZ is seriously lacking. For example, it is not known how LIPZ is homeostatically maintained under different physiological activities and whether zinc nutrition plays a role in influencing LIPZ concentration in the body. 1.2 Metallothionein M T is a low molecular weight (6000 - 7000) heavy metal-binding protein with high cysteine content (Davis et al. 1998). MTs are single chain proteins containing 61-62 amino acid residues and are structurally composed of two globular metal-binding a and (3 domains (Kagi, 1991; Haq et al., 2003). The twenty cysteine residues of MTs are conserved and function as heavy metal coordinators, which allow MTs to bind up to a total of seven zinc or up to twelve copper ions (Masters et al., 1994). The remarkably similar structure and high degree of homology of M T in different species imply its important biological functions (Haq et al., 2003). The most apparent structural motif of MTs from different species is the recurrence of Cys-X-Cys tripeptide sequences, where X stands for an amino acid residue other than cysteine (Kagi, 1991). The synthesis of M T usually occurs in organs responsible for absorption and excretion, such as the liver, intestine, pancreas, and kidney (Tapiero and Tew, 2003). Metallothionein is the major zinc-binding protein and the only known protein responsible for cellular zinc distribution (Ye et al., 2001; Zhou et al., 2002). 15 1.2.1 MT isoforms There are four known isoforms of MT: MT-1, MT-2, MT-3, and MT-4. MT-1 and MT-2 are the major isoforms with ubiquitous tissue distribution and particular abundance in the liver, pancreas, intestine, and kidney. These two proteins are thought to be functionally equivalent and highly inducible (Masters et al., 1994). MT-3 is expressed predominantly in the brain, although low expressions have been reported in the pancreas and intestine (Haq et al., 2003). Mice lacking MT-3 gene have low zinc concentrations in hippocampus and are more susceptible to seizures and neuron injuries (Erickson et al., 1997). MT-4 is found mainly in the epithelial cells in skin, tongue, upper stomach, and in the murine hair follicle (Lau et al. 1998; Davis et al. 2000; Schlake and Bochm, 2001; Haq et al., 2003; Tapiero and Tew, 2003). It is thought that MT-4 might play a role in zinc regulation during cellular differentiation in these tissues (Quaife et al., 1994). 1.2.2 Basal MT concentration The expression of MT is developmentally regulated. For example, MT rapidly declines to adult level within two to three weeks after birth in mice (Solaiman et al., 2001). In C57BL/6 mice, hepatic MT concentration reaches its peak at 3 days of age (279 ± 42 ug MT/g tissue), rapidly declines to 24 ± 2 ug MT/g by 14 days of age, and reaches adult level by 30 days of age (10 ± 0.2 ug MT/g; Lau and Cherian, 1998). In metallothionein transgenic (MT-TG) mice, similar pattern is observed in the change in MT during development. Hepatic MT concentrations are the highest from gestational day 20 (895 ± 125 ug MT/g) to 3 days of age (862 ± 135 ug MT/g) and reaches adult level by 21 days of age (250 ± 56 ug MT/g; Lau and Cherian, 1998). 16 In lactating mice, significant increases in hepatic M T concentration are observed in the liver on day 13 (~ 6-fold) and day 20 (~ 4-fold) of lactation compared to that of non-lactating mice, while hepatic M T concentration in the dam significantly decreases (5 - 7-fold) 5 days after weanling (Solaiman et al., 2001). Basal M T concentrations vary with species, gender, and type of tissues. The highest basal M T concentration is found in humans (~ 700 Hg MT/g liver) and dogs. Lower basal M T concentrations are found in monkeys and sheep (~ 200 u.g MT/g liver). The lowest basal M T concentrations, however, are found in mice, rats, and guinea-pigs (approximately between 2 to 10 u.g MT/g liver; Gibbs et al., 1985; Tapiero and Tew, 2003). Gender difference in M T concentration in mice has also been reported, as female M T -T G mice have 77% higher hepatic M T concentration than male M T - T G mice, while female MT-WT mice have 59% higher hepatic M T concentration than male MT-WT mice (Iszard et al., 1995). Of all the tissues examined in MT-WT mice, which included the brain, lungs, stomach, kidney, liver, spleen, testes, uterus, intestine, and pancreas, the highest M T concentration is found in the pancreas (~ 72 ug MT/g tissue) while the lowest is found in the lungs (~ 1.5 p.g MT/g tissue; Iszard et al., 1995). In M T - T G mice, the M T concentration found in thymus is 66.1 ± 7.92 ug MT/g tissue, which is 2.4 times higher than that in the MT-WT mice (27.0 ± 4.05 ug MT/g tissue; Deng et al., 1999). 1.2.3 Induction of MT M T is present at basal concentrations in all major mammalian organs, but its synthesis can be induced by various factors, such as heavy metals (e.g. Cd, Zn, Cu, Ag, and 1 7 Hg), tumor promoters (e.g. phorbol esters), hormones (e.g. glucagons and epinepherin), cytotoxic agents (e.g. chloroform), vitamins (e.g. ascorbic acid), antibiotics (e.g. cycloheximide), growth factors (e.g. IGF-1), inflammatory agents and cytokines (e.g. IL-6), antibiotics (e.g. cycloheximide) and physical stress (e.g. starvation, infection, ultraviolet radiation; Kag i , 1991; Davis and Cousins, 2000; Szczurek et al. , 2001; Kondoh et al. , 2003). For example, glucagon, the best candidate believed to induce M T during fasting, induces a higher M T accumulation in the liver and small intestine (72% and 50%, respectively) in glucagon-treated mice (Tran et al., 1998). Female C D - I mice injected with bacterial endotoxic-lipopolysaccharide (LPS) have a 10-20-fold increase in M T m R N A levels 4 hours post-injection (De et al., 1990). M T concentration can be induced by metals. Compared to 10 mg Zn/kg diet, 400 mg Zn/kg diet increases M T concentrations by as much as 100% in the small intestine in rats (Tran et al , 1999). Diethyl maleate ( D E M ) , which causes oxidative stress, also significantly induces hepatic M T m R N A in both mice and rats by as much as 37-fold and 25-fold, respectively (Bauman et al. , 1992). These factors regulate M T expression at the level o f gene transcription; however, the discrepancy between the level o f M T m R N A and M T protein suggests that M T synthesis is also regulated at the level o f translation. In cadmium-treated rats, although an increase in M T m R N A level in kidney is observed 4 - 6 h after the injection, no marked effect on M T protein was found (Vasconcelos et al., 2002). The inducibility o f M T is isoform-dependent. Available evidence has shown that events (e.g. zinc and cadmium treatments) inducing M T - 1 and M T - 2 do not generally enhance M T - 3 and M T - 4 expressions. Furthermore, the level of M T - 3 m R N A in mice is not affected by dexamethasone or endotoxin (lipopolysaccaride), two effective M T inducers in 18 the brain, while the levels of MT-1 and MT-2 mRNA are induced in the liver, pancreas, intestine, kidney, heart, lung, testis, and ovary in mice given 25 mM ZnSC»4 in their drinking water (Palmiter et al., 1992). However, the expression of MT isoforms is associated with other physiological activities, e.g. cell differentiation and proliferation (Bauman et al., 1992; Suhy et a l , 2003; Malaiyandi et al., 2004). Elevated levels of MT are found in rapidly growing tissues such as human tumors. For example, in HT-29 human colonic cancer cells, MT is synthesized rapidly and degraded during the progression of cell cycle (Nagel and Vallee, 1995). MT maximizes near G i / S transition, a period during which the cells prepare for DNA synthesis. During G O phase, however, only minimal amount of MT is observed (Nagel and Vallee, 1995). 1.2.4 Intracellular location of MT Intracellular location of MT depends on the status of proliferation. In adult liver or in confluent, non-stimulated cells, MTs are normally found in the cytoplasm (Schmidt and Beyersmann, 1999; Tran et al., 1999). MT is found in the nucleus during the late fetal and neonatal period in human and rat hepatocytes. Although the significance of nuclear M T is not known, it is thought that nuclear MT represents the transportation of zinc from cytoplasm into the nucleus to regulate the activities of zinc-finger containing transcription factors (Nartey et al., 1987; Davis et al., 1998; Lau and Cherian, 1998; Beyersmann and Haase, 2001). After stimulated by a hormone mixture (3-isobutyl-l-methylxanthine, dexamethasone, insulin, and 10% fetal calf serum), murine 3T3L1 fibroblasts start to translocate MT from cytosol into the nucleus. A decline in the proliferative cell fraction is then followed by a rapid decrease in nuclear MT (Schmidt and Beyersmann, 1999). Furthermore, upon 19 nitrosative stress induced by nitric oxide, M T in wild type murine aortic endothelial cells (WT MAEC) translocates to the nucleus to release zinc, which correlates with a nuclear appearance of labile zinc (Spahl et al., 2003). 1.2.5 Functions of M T The true physiological function of M T remains to be elucidated, but several roles of M T have been proposed. The proposed functions include detoxifying heavy metals (e.g. Cd and Hg), removing excess essential metals (e.g. Cu and Zn), regulating zinc and copper metabolism, and acting as an antioxidant by scavenging free radicals (e.g. superoxide anion, hydroxyl radical, and hypochlorous acid; Vallee and Falchuk 1993; Masters et al., 1994; Ogra and Suzuki, 2000). Generally speaking, M T can be considered as a toxin buffer that prevents heavy metal toxicity (Palmiter and Findley, 1995). High resistance to heavy metal toxicity in animals with M T overexpression further demonstrates MT's cytoprotective role (Hidalgo et al, 2001). Since the biosynthesis of M T can be stimulated by a large number of factors, the specific biological role of M T is difficult to identify. Even though M T seems to be multifunctional in the body, it is possible that M T is not a critical protein for normal development and reproduction (Masters et al., 1994; Davis and Cousins, 2000). This hypothesis is supported by the fact that M T knockout mice are generally in good health with no overt abnormalities (Masters et al., 1994; Lau and Cherian, 1998; Davis and Cousins, 2000). Furthermore, since cell lines failing to express M T still grow well and transfection of M T genes into these cells does not improve growth, M T might not be essential for the synthesis or function of metalloenzymes or other metalloproteins (Masters et al., 1994). 20 1.3 Zinc Homeostasis Homeostasis can be defined as the body's ability to maintain a relatively constant internal state with varying external conditions (King et al., 2000). Zinc is not stored in the body at significant quantities; therefore, maintaining an adequate and stable state of cellular zinc is essential for maintaining normal functions in the body (King et al., 2000; Tapiero and Tew, 2003). Unlike iron, which can be stored in the body as ferritin, there is no true zinc storage proteins identified. To date, M T is the only protein that has been implicated in cellular zinc storage (Kagi and Schaffer, 1988). During zinc deficiency, body zinc reserves from M T can be donated to target apometalloenzymes, e.g. zinc-finger transcription factors, in order to maintain regular body functions (Kagi and Schaffer, 1988; Davis and Cousins, 2000). In cultured adult rat hepatocytes, when zinc (100 uM) is removed from the medium, M T turnover rate is almost 3 times faster than that in the hepatocytes grown in regular medium. It is thought that once zinc reserves are exhausted, some zinc metalloenzymes will start to degrade. The degradation of zinc metalloenzymes leads to the release of zinc from those metalloenzymes that hold zinc less securely, e.g. carboxypeptidase A (DiSilverstro et al., 1983). On the other hand, excessive zinc interferes with other metal-dependent processes and/or inhibits other proteins (Maret, 2000). For example, copper-deficiency anemia secondary to excess zinc (e.g. 20 mg for 7 months or 2,000 mg for 3 months) was first reported in 1977 in humans (Simon et al., 1988; Forman et al., 1990). Excess dietary zinc induces the synthesis of M T , which also binds to copper. However, since M T has much higher binding affinity for copper (~ 3.2 x 10 1 7 M " 1 at pH 7.4) than for zinc (~ 3.2 x 10 1 3 M " 1 at pH 7.4), the newly synthesized thionein induced by zinc intake binds to copper and prevents the copper from entering the plasma (Jacob et a l , 1998; Milne, 1998). Clearly, zinc 21 homeostasis is compromised during periods of inadequate- or excessive-zinc intake. Adverse clinical symptoms may develop as a consequence. Under normal physiological conditions and at adequate-zinc intake, zinc homeostasis is maintained. To achieve zinc homeostasis, three different mechanisms are thought to be involved: absorption and excretion, regulation of M T , and regulation of zinc transporters. 1.3.1 Absorption and excretion The gastrointestinal tract is one of the major sites for zinc homeostasis. One of the zinc homeostatic mechanisms involves the regulation of zinc absorption and excretion in response to zinc intake levels. Techniques using the three stable isotopes of zinc ( 6 7Zn, 6 8 Z n , and 7 0 Zn) provide information on how effectively the intestine absorbs exogenous dietary zinc and conserves endogenous zinc. Zinc is absorbed from the small intestine, primarily the duodenum and jejunum. The highest zinc absorption rate is found within jejunum (Cousins, 1997; Coyle et al., 2000). Zinc absorption responds rapidly to alterations in dietary zinc. In general, absorption efficiency increases at low-zinc intake, while absorption efficiency decreases at high-zinc intake. At the same time, the amount of endogenous zinc excretion increases/decreases as dietary zinc intake increases/decreases (King et a l , 2000; Lonnerdal, 2000; Krebs et al., 2003). For example, zinc supplementation of 22 mg Zn/d for 28 days decreases fractional zinc absorption from 22 to 8% and increases urinary zinc concentration in Korean women (Kim et al., 2004). Acute zinc depletion for 5 weeks (0.23 mg Zn/d) increases fractional zinc absorption from 26% to essentially 100% and causes a 96% decrease in urinary and fecal zinc excretion in men with an average baseline zinc intake of 12.2 mg Zn/d (King et al., 2001). 22 Other than zinc intake, physiological conditions (e.g. lactation) and other food components (e.g. phytate and iron) also play a role in regulating the absorption and excretion of zinc. For example, compared to themselves before conception, lactating women have a 2-fold increase in the fractional zinc absorption (Fung et a l , 1997). Despite the higher fractional zinc absorption in lactating women, the fecal and urinary zinc excretion is not significantly increased, indicating higher zinc conservation (Sian et al., 2002). Women consuming a lactoovovegetarian (molar ratio of phytate to zinc =14) diet for 8 weeks have a 35% reduction in zinc absorption compared to those who consume a nonvegetarian diet (molar ratio of phytate to zinc = 5), although zinc balance is maintained with both diets (Hunt et al., 1998).' A single 60-mg dose of ferrous sulfate also reduces fractional zinc absorption by 5% during 7 - 9 wk of lactation (Chung et al., 2002). Overall, although various factors can influence the absorption and excretion of zinc, the combined action of modifying intestinal zinc absorption and endogenous zinc excretion takes part in regulating zinc homeostasis. 1.3.2 Zinc and M T M T is the major zinc-binding protein, and zinc is one of the most potent inducers of M T synthesis. One molecule of M T is capable of binding seven atoms of zinc (Davis et al., 1998). As previously mentioned, cytoplasmic M T can translocate into the nucleus in proliferating cells. Intracellular zinc accumulation is observed in proliferating cells. For example, in the wounded epidermis of mice, a gradual increase in M T mRNA level followed by zinc accumulation in wounded epidermis is observed (Iwata et al., 1999). Therefore, it is proposed that the translocation of M T represents the transportation of zinc by M T from 23 cytoplasm into the nucleus, where zinc is required by enzymes for DNA synthesis (e.g. D N A polymerase) and zinc-finger proteins (Lau and Cherian, 1998; Beyersmann and Haase, 2001). Redistribution of M T to the cytoplasm occurs once DNA synthesis is initiated, i.e. enter S-phase of the cell division cycle (Lau and Cherian, 1998). As an intracellular zinc-binding protein, M T may regulate intracellular free zinc levels (Kondo et al., 1997; Suhy et al., 1999). Thionein (T), the apo-MT, is a potent zinc acceptor. However, thionein does not necessarily act as a strong chelating agent towards enzymes (Jacob et al., 1998). Moreover, despite MT's high metal binding constant (ATzn = 3.2 x 1013 M" 1 at pH 7.4), it can transfer zinc to apometalloenzymes with lower zinc binding affinity (Kagi and Schaffer, 1988; Davis and Cousins, 2000). In rats, radioactive zinc included in zinc-deficient diet quickly appears as 6 5 Zn-MT. Instead of reaching a plateau, 6 5 Z n - M T concentration fluctuates. It markedly decreases between 10 and 12 h after zinc feeding, followed by a dramatic increase at 18 h. The decrease in 6 5 Z n - M T between 10 and 12 h suggests a donation of zinc from M T to other intracellular ligands, while the increase at 18 h suggests a newly formed 6 5 Z n - M T complex (McCormick et al., 1981). The acceptance of zinc by thionein and the donation of zinc by M T are more effective in the presence of other cellular ligands or chelating agents (e.g. glutathione; Jacob et al., 1998). a) Dietary zinc intake and MT. One of the proposed roles for M T is to regulate zinc homeostasis. At adequate-zinc intake, M T synthesis is low (Davis et al., 1998). In mice, basal M T concentration ranges from approximately 1.5 ug/g lungs to 72 ug/g pancreas, depending on the tissue type (Iszard et al., 1995). However, the absence of M T staining in surface epithelial cells in response to severe dietary zinc deficiency (< 1 mg Zn/kg diet) and 24 the presence of strong M T staining after zinc repletion (30 mg Zn/kg diet) indicate that the synthesis of M T can be stimulated by dietary zinc intake (Szczurek et al., 2001). The elevation of zinc in growing medium increases the concentration of zinc in the cellular zinc pool, which enhances the binding of zinc to metal-responsive transcription factor (MTF-1). MTF-1 is a metal-regulatory protein generally located in the cytoplasm. Upon sensing the increase in intracellular free zinc, it rapidly (within 30 min after 100 uM zinc treatment in Hepa cells) translocates into the nucleus to activate metal response element (MRE; Smirnova et al., 2000). M R E can then up-regulate the synthesis of M T (Davis and Cousins, 2000). The binding activity of MTF-1 varies in response to cellular free zinc concentrations (Bittel et al., 1998), and MTF-1 activation by zinc is reversible (Andrews, 2000). Studies have shown that the expression of both hepatic and intestinal MT-1 mRNA in male rats is significantly elevated by zinc supplementation (180 mg Zn/kg diet), and depressed by zinc restriction (5 mg Zn/kg diet; McMahon and Cousins, 1998). Furthermore, the rapid increase of zinc in the nucleus after high-zinc diet also suggests a direct link between M T gene expression and dietary zinc (Cousins and Lee-Ambrose, 1992). b) MT adaptation to dietary zinc intake Responses of M T to changes in dietary zinc intake can occur within the first few hours after zinc intake (Szczurek et al., 2001). For example, 6 h after feeding a zinc-supplemented diet (383 ug ZnCb/ml diet), male rats have a significant increase in hepatic M T concentration. The concentration of hepatic M T in the rats peaks at 12 h after feeding and remains unchanged through 18 h (McCormick et al., 1981). By incorporating 6 5 Z n into the liquid diet, McCormick et al. (1981) also found that the rate of M T synthesis increases (4.5-fold) 10 h after zinc intake. A rapid appearance of 25 w Z n is observed in the liver with M T (Mccormick et al., 1981). The induction of M T synthesis at high-zinc intake may only be the short-term strategy for coping with high-zinc intake, as M T synthesis in the intestine peaks on day 3 and starts to decline to normal concentration by day 14 after high-zinc intake in 8-week-old Sprague-Dawley rats fed 350 mg Zn/kg diet for 42 days (Reeves, 1995). In M T - T G mice and its genetic control mice, hepatic M T concentration increases from baseline (Od) 9 days (MT-TG: 48%; control: 60%) or 18 days (MT-TG: 70%; control: 55%) after intake of zinc-deficient diet (0.5 - 1.5 ng /g diet). Hepatic M T concentrations in both M T - T G and the control mice decrease after 27 days of low-zinc intake. The values return to day 9 value although they are still higher than that at baseline (Dalton et al., 1996). Therefore, it is suggested that during zinc deficiency, MT-bound zinc represents a bioavailable pool of zinc, and the concentration of M T decreases as the need for zinc increases. Zinc absorption is depressed after ingesting zinc-rich diets, and the decline in absorption has been found to be correlated with an increase in M T synthesis (Cousins, 1997; Davis et al., 1998). The observed relationship between M T synthesis and zinc absorption has envisioned M T as an expandable zinc pool within the enterocytes (Cousins, 1997). M T can serve as an intracellular buffer of zinc movement by functioning as a sink that holds zinc in the intestine, which allows more opportunity for the transportation of zinc back into the lumen (Davis et al., 1998; Cousins, 1997). Overall, the expression of M T is influenced by dietary zinc intake, and zinc absorption is correlated with M T synthesis. Even though at this point, not all MT's functions are well understood, MT's role in regulating zinc homeostasis has been documented by many studies. 26 1.3.3 Z i n c t r a n s p o r t e r s Absorption of zinc requires the involvement of various transporters to facilitate the influx and efflux of zinc from cells, as ionic zinc (Zn 2 +) is highly charged and cannot cross biological membranes by passive diffusion (Davis et al., 1998; McMahon and Cousins, 1998; McMahon and Cousins, 1998). ZnT. (zinc transporter) or CDF (cation diffusion facilitator) is a family of mammalian zinc transporters, which export zinc ions from cells or into cytosolic compartments (Cousins and McMahon, 2000; Harris, 2002). A total of 9 ZnTs (ZnT-1 to ZnT-9) have been cloned to date. ZIP (Zrt-and Irt-like proteins) is another family of transporters. The ZEP family is now considered as the best candidate for cellular zinc influx (Wardrop and Richardson, 1999; Harris, 2002). Another metal transporter, Nramp2, also known as DCT-1 (divalent cation transporter 1), D M T (divalent metal transporter), and SLC11A2, is also considered to have the potential for zinc influx activity (Jabado et al., 2002; Kambe et al., 2003). These zinc transporters differ in tissue specificity, cellular location, outward or inward movement, regulated or constitutive expression, and sensitivity to zinc (Harris, 2002; Kambe et al., 2003; Figure 1-1). 27 ZnT-8 ZnT-1 - Pancreas - Liver - All tissues ZnT-4 - Prostate - Liver - Jejunum - Kidney - Bone marrow - Other tissues - Duodenum N r a m p 2 - Uterus - Skin - Mammary gland - Brain - Liver - Intestine * T G N = Trans-Golgi Network Figure 1-1. Zinc transporters (Adopted from Liuzzi and Cousins, 2004 with modifications.) a) Zinc exporters. ZnT-1 mRNA is expressed ubiquitously in the body, with placenta, kidney, adipose, and intestine having the highest levels (Harris, 2002). ZnT-1 is localized at the basolateral membrane of enterocytes and renal tubular cells where it is configured to release absorbed zinc across the membrane into capillaries, or into renal tubules of the kidney (Harris, 2002; Liuzzi and Cousins, 2004). In the intestine, ZnT-1 is most abundant near the basolateral membrane of duodenum and jejunum (Cousins et al., 2003). Cells that over-express ZnT-1 are much more resistant to high concentrations of extracellular zinc and have lower intracellular zinc levels (McMahon and Cousins, 1998). The introduction of ZnT-1 into zinc-sensitive mutant BHK (baby hamster kidney) cells restores normal zinc resistance, as the maximal zinc concentration compatible with cell growth increases from 30 to 100 uM 28 (Palmiter and Findley, 1995). These observations lead to the hypothesis that ZnT-1 expression can be a defense mechanism during high-zinc intake by exporting zinc out of the cell (McMahon and Cousins, 1998; Cousins and McMahon, 2000). However, the growth of BHK cells is retarded when intracellular zinc exceeds ~ 300 uM, indicating ZnT-1 might be fully active and cannot effectively prevent zinc toxicity at this concentration or above (Palmiter and Findley, 1995). ZnT-2 is primarily found in the intestine, seminal vesicles, placenta, kidney, and testis (Cousins and McMahon, 2000; Huang et a l , 2002). Cells transfected with ZnT-2 gene not only survive at much higher concentrations of extracellular zinc, but also exhibit a highly vacuolated appearance (McMahon and Cousins, 1998). Therefore, it is thought that ZnT-2 delivers excessive zinc into intracellular compartments. The expression of ZnT-3 has been reported in the brain and testis (McMahon and Cousins, 1998). ZnT-3 is located at the vesicular membranes and is involved in transporting zinc from the cytoplasm into vesicles (Huang et al., 2002). The actual function of ZnT-3 is not clear; however, it is likely that ZnT-3 has a role in neurodegenerative function and spermatogenesis (Cousins and McMahon, 2000; Tapiero and Tew, 2003). ZnT-4 is present in the brain, mammary gland, liver, lung, kidney, spleen, heart, and small intestine (Cousins and McMahon, 2000; Huang et al., 2002). ZnT-4 restores zinc resistance in a yeast strain (Azrcl) that has a defect in a vacuolar zinc transporter, indicating that ZnT-4 is involved in exporting cytoplasmic zinc into vacuoles (McMahon and Cousins, 1998; Huang et al., 2002). ZnT-4 is also found to play a critical role in depositing cytoplasmic zinc into secretory vesicles in lactating mammary glands (Huang et al., 2002). A single point mutation in ZnT-4 can result in lethal (Im) milk syndrome in mice (Tapiero 29 and Tew, 2003). The markedly less zinc secretion into breast milk leads to zinc deficiency in pups and death before weanling (Lee et al., 1992). The mutant pups, however, can survive if fostered to a normal dam, or supplemented with zinc (Ackland and Mercer, 1992). Recently, ZnT-5, -6 and -7 have been identified. Human ZnT-5 (hZnT-5) is only found recently in human pancreatic B cells (Kambe et al., 2002). The role of ZnT-5 is not well understood; however, it is suggested that ZnT-5 plays an important role in transporting zinc into secretory granules in pancreatic cells (Kambe et al., 2002). The localization of ZnT-6 is not known, but studies using yeast strains have shown that ZnT-6 functions as a zinc transporter responsible for relocating cytoplasmic zinc into the trans.Golgi network (TGN) as well as the vesicular compartment (Huang et al., 2002). ZnT-7 expression is restricted to lung and small intestine (Kirschke and Huang, 2003). Accumulation of zinc in the Golgi apparatus of the ZnT-7 over-expressing Chinese hamster ovary (CHO) cells suggests that ZnT-7 facilitates zinc transportation from cytoplasm into the Golgi apparatus (Kirschke and Huang, 2003). More recently, a human ZnT-8 sequence has been reported. It is found to share a 42% homology with ZnT-3. Although ZnT-8 transcripts are detected in the liver and pancreas in mice, its characteristics are yet to be found out (Liuzzi and Cousins, 2004). The putative ZnT-9 reference sequence (NM 006345) encodes a cation efflux motif. However, instead of associating with plasma membrane or microsomes, ZnT-9 has been found in the cytoplasm and nuclear fraction of MRC-5 cells (human embryonic lung cell; Sim and Chow, 1999). The role of ZnT-9 is currently not yet established, and no evidence related to nutrition is found (Liuzzi and Cousins, 2004). 30 b) Zinc importers. ZIP (ZRT-1, IRT-l-like protein) is a family of transporters with structural and functional analogy to both ZRT-1 and IRT-l-like proteins (Harris, 2002). Thus far, fourteen ZIP proteins have been identified in humans (hZIPl - hZIP14; Wang et al., 2004). The ZIP family is originally thought to be iron transporters, but it is now found to also transport Z n 2 + , M n 2 + , C d 2 + , and other divalent cations (Harris, 2002). ZIPl has a widespread tissue distribution, while ZIP2 and ZIP3 are more abundant in tissues like the spleen, bone marrow and small intestine. Mouse ZBP4 and ZIP5 are co-expressed in the liver, intestine, kidney, pancreas, and embryonic yolk sac (Dufner-Beattie et a l , 2004; Wang et al., 2004). In M D C K (Madin-Darby canine kidney) cells, mZrP4 primarily localizes to the apical plasma membrane, while the mZIP5 is found only on the basolateral surface (Wang et al., 2004). Although it is suggested that mammalian ZIP genes are integral components of the zinc homeostatic mechanism, the mechanisms are not clear (Dufner-Beattie et a l , 2003). For example, a study investigates the relationship between dietary zinc and mouse ZIP mRNA levels found that mouse ZIPl, ZIP2, and ZEP3 mRNA levels are not regulated by dietary zinc intake (Dufner-Beattie et al., 2003). It is therefore suggested that mouse ZIPl, ZIP2, and ZTP3 proteins are possibly regulated translational and/or post-translationally in response to zinc intake (Dufner-Beattie et al., 2003). However, the protein level of mZIP4 at the plasma membrane in HEK293 (human embryonic kidney) cells is found to increase in response to zinc deficiency (Dulbecco's modified Eagle's medium with 10% fetal bovine serum treated with Chelex 100), and the increase in mZIP4 protein is associated with an increase in zinc uptake (Kim et al., 2004). The expression of mZIP5 protein is, however, not altered by zinc deficiency. In HEK 293 cells, zinc repletion after zinc deficiency does not down-regulate the 31 activity of mZIP5 (Wang et al., 2004). Furthermore, in mice fed zinc-deficient diet (1 mg Zn/kg diet), intestinal ZIP5 mRNA level is unaffected, whereas intestinal ZB?4 mRNA level increases 24 h after zinc-deficient diet and continues to increase for five days (Dufner-Beattie et al., 2004). During zinc deficiency, ZIP5 is removed from the cell surface and internalized, while ZEP4 is induced and recruited to the apical surface of enterocytes (Dufner-Beattie et al., 2004). Although ZIP1, ZIP2, and ZIP3 are not affected by zinc intake in mice, Z1P4 and ZIP5 seem to work cooperatively in the intestine to import zinc during zinc deficiency. Nramp2, located on the plasma membrane, is expressed in a wide variety of tissues (Wardrop and Richardson, 1999). It is essential for iron absorption and is up-regulated by iron deficiency (Wardrop and Richardson, 1999; Cousins and McMahon, 2000). Numerous studies have shown enhanced zinc absorption at low-iron intake (Cousins and McMahon, 2000). However, the direct relationship between dietary zinc and Nramp2 has not been investigated. c) Effects of zinc intake on zinc transporters. The effects of dietary zinc intake on the expression of ZnTs are not yet well studied. Limited evidence suggests that although the expression of ZnTs can be influenced by dietary zinc intake, ZnTs are generally not very responsive to zinc intake. In the intestine, ZnT-1 mRNA levels elevate significantly in animals fed zinc-supplemented diet (e.g. 180 mg Zn/kg diet); however, ZnT-1 mRNA level in the intestine of zinc-restricted animals (e.g. 5 mg Zn/kg diet) is not altered significantly relative to the zinc-adequate animals (e.g. 30 mg Zn/kg diet; McMahon and Cousins, 1998). Moreover, even though both the expressions of ZnT-1 and M T are affected by dietary zinc intake, the expression of ZnT-1 does not depend on the level of M T and the animal's 32 capacity to produce MT. The magnitude of ZnT-1 mRNA in response to oral zinc dosing (0.5 mmol Zn/kg body weight) is similar between M T - T G and M T - K O mice compared to their respective controls (McMahon and Cousins, 1998; Cousins and McMahon, 2000). Therefore, M T does not seem to affect the abundance of ZnT-1 mRNA. In M T - W T mice, ZnT-1 mRNA level in the liver is not affected by 2 weeks of low-zinc (< 1 mg Zn/kg diet) or high-zinc intake (180 mg Zn/kg diet) in rats. The responsiveness of ZnT-2 to dietary zinc intake is greater than that of ZnT-1. ZnT-2 mRNA level is more closely related to dietary zinc intake. For example, ZnT-2 mRNA level is non-detectable in the liver in rats during zinc deficiency (< 1 mg Zn/kg diet), but it is significantly elevated in animals fed zinc-supplemented diet (180 mg Zn/kg diet) (Cousins and MaMahon, 2000; Liuzzi et al., 2001). ZnT-1 mRNA level in the small intestine in rats is not lowered during zinc deficiency (< lmg Zn/kg diet) compared to that at adequate-zinc intake (30 mg Zn/kg diet); however, ZnT-2 mRNA level is lower during zinc deficiency (< 1 mg Zn/kg diet) and higher during zinc supplementation (180 mg Zn/kg diet) compared to that at adequate-zinc intake (Liuzzi et al., 2001). In the kidney, however, only high-zinc intake significantly increases the levels of both ZnT-1 and ZnT-2 mRNA (Liuzzi et al., 2001). Various studies have reported a lack of influence in ZnT-4 mRNA levels in the liver, kidney, and small intestine by dietary zinc intake (Liuzzi et a l , 2001; Harris, 2002). Overall, our understanding of the relationship between dietary zinc intake and the expressions of zinc transporters, and the contribution of zinc transporters to zinc homeostasis are in its infancy. Available evidence has shown that the expressions of ZnT-1, -2, and -4 appear to be influenced by dietary zinc intake. Thus far, the responses of ZnT-3, -5, -6, -7,-33 8,-9, ZIP, and DCT-1 to dietary zinc intake have not been established. Moreover, only a limited number of studies suggest that ZnT-1 and ZnT-2 contribute to zinc homeostasis. 1.4 Dietary zinc, M T , and L I P Z Thionein synthesis is stimulated by high dietary zinc intakes and suppressed by low-zinc intakes. The newly synthesized thionein binds to zinc to form MT. The formation of M T reduces the concentration of free intracellular ionic zinc, preventing potential zinc toxicity. Evidence also suggests that under conditions of zinc deprivation, M T is degraded to release zinc to support metabolic processes (Tran et al., 1999; Langmade et al., 2000). The relationship between dietary zinc and the abundance of LEPZ has not yet been well studied. An in vitro study using 3T3 cells first demonstrated a positive correlation between zinc nutrition and the abundance of LIPZ (Paski & Xu, 2001). Supplementation of zinc to zinc-depleted medium increases the abundance of LIPZ (Paski & Xu, 2001). In animals, the labile intracellular zinc is referred to as the "histochemically reactive zinc." However, study examining the relationship between dietary zinc intake and histochemically reactive zinc in animals is generally lacking. Recently, a correlation between the abundance of intestinal histochemically reactive zinc and dietary zinc intake was demonstrated by Paski et al. (2003), in which dietary zinc deficiency results in a lower abundance of intestinal histochemically reactive zinc in rats, while zinc supplementation increases the abundance of the intestinal histochemically reactive zinc. Since the majority of intracellular zinc is bound to macromolecules such as proteins, LIPZ logically provides zinc that is more readily available for participating in zinc-requiring metabolic activities. Lower abundance of LIPZ might impede metabolic activities that 34 require zinc, while excessive abundance of LIPZ could cause zinc toxicity. Therefore, it is important to maintain LIPZ homeostasis. Thus far, our knowledge about the regulation for maintaining LIPZ homeostasis is severely limited. Metallothionein, together with zinc transporters, seem to be good candidates in maintaining LIPZ homeostasis, as M T releases zinc during zinc deficiency and sequesters zinc during high zinc situations. Paski et al. (2003) have recently shown a possible relationship between M T and histochemically reactive zinc. Organs with higher M T concentrations (e.g. liver) have lower histochemically reactive zinc abundance than those with lower M T concentrations (e.g. intestine), even though their total zinc concentrations are similar. Molecular-mass profile of the liver in rats also suggests that although TSQ-reactive zinc and MT-bound zinc represent two different pools of zinc, the two pools are interrelated (Paski et al., 2003). However, the molecular-mass profile of the liver requires tissue homogenization, but homogenization can introduce possible artifact. Therefore, the role of M T in LIPZ regulation still remains unclear. 2. Hypothesis Based on the available information, we hypothesize that the abundance of histochemically reactive zinc in mice will be homeostatically regulated through the interplay between M T and zinc transporters. Upon entering the cell, zinc contributes to the histochemically reactive zinc pool. When cellular zinc uptake is high, such as in the case of high-zinc intake, zinc from the histochemically reactive zinc pool stimulates the synthesis of thionein, which becomes M T upon binding to zinc. If the abundance of histochemically reactive zinc pool is low, such as in the case of low-zinc intake, M T donates its zinc back to the histochemically reactive zinc pool. Meanwhile, the expression of zinc transporters is 35 either increased or decreased according to zinc intake levels. For example, at high-zinc intake, the expression of zinc importers might be down-regulated while the expression of zinc exporters might be upregulated to prevent excessive accumulation of histochemically reactive zinc. At low-zinc intake, this process might be reversed to acquire as much zinc as possible. Therefore, we hypothesize that M T and zinc transporters work in concert to maintain adequate pool of histochemically reactive zinc (Figure 1-2). Dietary Zn Zinc MRE = Metal Response Element Histochemically reactive zinc Figure 1-2. Rationale 36 3. Objective The overall objective of this study was to investigate the role of M T and zinc transporters in the homeostatic regulation of histochemically reactive zinc in response to various zinc intakes in mice. The specific objectives of this study were: 1) to examine the effect of dietary zinc intake and treatment duration on the abundance of histochemically reactive zinc in M T - W T mice; 2) to examine the effect of dietary zinc intake and treatment duration on the abundance of ZnT-1, ZnT-2, ZnT-4, and Nramp2 mRNA levels in MT-WT mice; 3) to examine the effect of M T elimination on the abundance of histochemically reactive zinc using M T - K O mice; and 4) to examine the effect of M T elimination on the abundance of ZnT-1, ZnT-2, ZnT-4, and Nramp2 mRNA levels using M T - K O mice. 37 C H A P T E R n M E T A L L O T H I O N E I N , N O T ZINC T R A N S P O R T E R S , IS I M P O R T A N T T O T H E HOMEOSTASIS O F H I S T O C H E M I C A L L Y R E A C T T V E ZINC IN M I C E INTRODUCTION Zinc, a trace mineral, is present in all organs, tissues and body fluids in animals and humans (Cousins, 1997). About 99 % of the body zinc is present intracellularly (Vallee, 1993; Cousins, 1997). Cellular zinc can also be categorized based on its dynamics: the essentially non-exchangeable pool of zinc and the labile pool of zinc. The majority (~ 90%) of the intracellular zinc is found in the non-exchangeable pool of zinc, which consists of zinc that is tightly bound within the tertiary protein structure of zinc metalloenzymes (Miller et al., 1994; Truong-Tran et al., 2000; Truong-Tran et al., 2003) and zinc-finger proteins (Klug and Schwabe, 1995). The remaining portion of the intracellular zinc is free zinc ions and zinc that are loosely bound to macromolecules. Collectively, this pool of zinc is known as the labile intracellular pool of zinc (LIPZ). The actual size and the homeostatic control of LIPZ are presently not known, although the concentration of intracellular free ionic zinc is estimated to be in the picomolar or nanomolar range (Maret, 2000; Malaiyandi et al., 2004). It has been suggested that some of the intracellular ionic zinc is "stored" in subcellular vesicles, which are referred to as "zincosomes" (Truong-Tran et al., 2000; Beyersmann and Haase, 2001). However, zincosomes are not well defined experimentally and their characteristics are essentially unknown thus far. LIPZ has been shown to be critical to metabolic events such as cell proliferation and apoptosis. However, our knowledge in the homeostatic control of LIPZ is seriously lacking. 38 Metallothionein is a low molecular weight heavy metal-binding protein with high cysteine content (Davis et al. 1998). M T is the major zinc-binding protein and plays an important role in cellular zinc distribution (Ye et al., 2001; Zhou et al., 2002). There are four known isoforms of MT: MT-1, MT-2, MT-3, and MT-4. MT-1 and MT-2 are the major isoforms, which have ubiquitous tissue distribution with particular abundance in the liver, pancreas, intestine, and kidney. These two isoforms of M T are thought to be functionally equivalent (Masters et al., 1994). MT-3 is expressed predominantly in the brain, although low expressions have been reported in the pancreas and intestine (Haq et al., 2003). Mice lacking MT-3 gene have low zinc concentrations in hippocampus and are more susceptible to seizures and neuron injuries (Erickson et al., 1997). MT-4 is found mainly in the epithelial cells in skin, tongue, upper stomach, and in the murine hair follicle (Lau et al. 1998; Davis et al. 2000; Schlake and Bochm, 2001; Haq et al., 2003; Tapiero and Tew, 2003). The expression of M T is developmentally regulated, as M T rapidly declines to adult levels within two to three weeks after birth in mice (Solaiman et al., 2001). Basal M T concentration varies with tissue types. In mice, basal M T concentration is the highest in the pancreas and lowest in the lungs (Iszard et al., 1995). M T is present at basal levels in all major mammalian organs, but its synthesis can be induced by various factors, including zinc (Kagi, 1991; Davis arid Cousins, 2000; Szczurek et al., 2001; Kondoh et al., 2003). However, the inducibility of M T is isoform-independent. Dietary zinc induces the synthesis of MT-1 and MT-2 but not MT-3 and MT-4. Zinc is not stored in the body at significant quantity; therefore, maintaining an adequate and stable state of cellular zinc is essential for maintaining normal functions in the body (King et al., 2000; Tapiero and Tew, 2003). To date, M T is the only protein that has 39 been implicated in cellular zinc storage (Kagi and Schaffer, 1988). Under normal physiological conditions and at adequate-zinc intake, zinc homeostasis is primarily maintained through the regulation of absorption and/or excretion, the synthesis of M T , and zinc transporters. Zinc transporters are required to facilitate the influx and efflux of zinc from cells, as ionic zinc (Zn 2 +) is highly charged and cannot cross biological membranes by passive diffusion (Davis et al., 1998; McMahon and Cousins, 1998; McMahon and Cousins, 1998). ZnT (zinc transporter) or CDF (cation diffusion facilitator) is a family of mammalian zinc transporters, which export zinc ions from cells or into cytosolic compartments (Cousins and McMahon, 2000; Harris, 2002). A total of 9 ZnTs have been cloned to date. ZIP (Zrt-and Irt-like proteins) is another family of transporters. The ZIP family is now considered the best candidate for cellular zinc influx (Wardrop and Richardson, 1999; Harris, 2002). Another metal transporter, Nramp2, also known as D C T 1 (divalent cation transporter 1, DCT-1), D M T (divalent metal transporter), and SLC11A2, is also considered to have the potential for zinc influx activity (Jabado et al., 2002; Kambe et al., 2003). These zinc transporters differ in tissue specificity, cellular location, outward or inward movement, regulated or constitutive expression, and sensitivity to zinc (Harris, 2002; Kambe et a l , 2003). However, our knowledge in zinc transporters is still in its infancy. For example, although zinc transporters have been proposed to play an important role in zinc homeostasis, influence of dietary zinc intake on the expression and synthesis of these zinc transporters is largely unclear. The abundance of LIPZ can be influenced by zinc nutrition. In 3T3 cells, the abundance of LIPZ is reduced by the depletion of zinc in culture medium and increased by the supplementation of zinc in culture medium (Paski & Xu, 2001). This association 40 between zinc nutrition and LIPZ abundance has also been demonstrated in rats. In animals, LIPZ is referred to as histochemically reactive zinc. Paski et al. (2003) have shown that dietary zinc deficiency results in a lower abundance of intestinal histochemically reactive zinc in rats, while zinc supplementation increases the abundance of intestinal histochemically reactive zinc. Paski et al. (2003) also suggest that a portion of MT-bound zinc is part of the histochemically reactive zinc pool. However, the homeostatic regulation of histochemically reactive zinc is not known. Based on MT's and zinc transporters' roles in zinc homeostasis, we hypothesize that M T and zinc transporters are also important in the homeostatic regulation of histochemically reactive zinc. The objectives of this study were to investigate: 1) the effect of zinc intake and treatment duration on the abundance of histochemically reactive zinc and zinc transporters; and 2) the effect of M T elimination on the abundance of histochemically reactive zinc and zinc transporters using M T - K O mice. M A T E R I A L S A N D M E T H O D S Diets Three diets; adequate-zinc (30 mg Zn/kg diet), low-zinc (2 mg Zn/kg diet), and high-zinc (150 mg Zn/kg diet) diets were prepared based on the modified egg-white based AIN-93G diet (Reeves et al., 1993). Compositions of the diets are given in Appendix I. The adequate-zinc diet was the control diet (Tallman and Taylor, 2003). The low-zinc diet was designed based on a preliminary study to develop marginal zinc deficiency in mice, while the high-zinc diet was designed to mimic zinc supplementation. These diets were in powder form. When mice are fed diets in powder form, feed spilling is common, which results in serious errors in assessing feed intake. To minimize feed spilling, thick pasty diets were 41 prepared by mixing the powder diets with double-deionized water (20 ml double deionized water/100 g powder diet; Tallman and Taylor, 2003). Animals, dietary treatments, and animal care Metallothionein-wild type (MT-WT) mice: twenty-one-day old male MT-WT mice (129S1) were purchased from the Jackson Laboratory (Bar Harbor, Maine, USA). After two days of acclimatization, the twenty-three-day old mice were randomly assigned into one of the three zinc treatment groups: low-zinc (Zn2; n = 7), adequate-zinc (Zn30; n = 6), or high-zinc (Znl50; n = 6) group with an average initial body weight of 9.5 g (6.7 g -12.9 g). The mice were maintained on their assigned diet for 2, 4, 7, or 21 days. The time points were chosen to investigate both the short-term and long-term responses of histochemically reactive zinc and zinc transporter to dietary zinc intake. All mice had free access to their assigned diet and double-deionized water throughout the feeding trial. The glass feeding jars and high-density polyethylene water bottles used were thoroughly cleaned, treated with ethylenediamine tetraacetate (EDTA) solution, and rinsed with double-deionized water to prevent zinc cross contamination. In addition, six mice (twenty-one days old) were fed laboratory rodent chow with free access to double- deionized water. After two days on laboratory rodent chow, tissues were removed from these mice to obtain baseline data. Feed intakes were recorded every other day and body weight was obtained weekly. The mice were housed in stainless steel cages in a temperature- (~ 21°C) and humidity-regulated room with a 12-hour light-dark cycle. Metallothionein-knockout (MT-KO) mice: twenty-one-day old male MT-KO mice (129S7) were purchased from the Jackson Laboratory (Bar Harbor, Maine, USA). After two 42 days of acclimatization, the twenty-three-day old mice were randomly assigned into one of the three zinc treatment groups (n = 5-6 mice/group): low-zinc (Zn2), adequate-zinc (Zn30), or high-zinc (Znl50) group with an average initial body weight of 9.0 g (6.9 g - 10.3 g). The mice were maintained on their assigned diet for 4 days. A l l mice had free access to their assigned diet and double-deionized water throughout the feeding trial. In addition, six mice (twenty-one days old) were fed laboratory rodent chow with free access to double-deionized water. After two days on laboratory rodent chow, tissues were removed from these mice to obtain baseline data. Feed intakes were recorded every other day and body weight was obtained twice: at the beginning and the end of the feeding trial. The mice were housed in the same conditions as described above. Tissue collection and preparation At the end of each feeding trial, bone (both left and right femur and tibia), the liver, and small intestine were removed for analyses while the mice were under anesthesia. The liver and small intestine were selected for the study because both the liver and small intestine were important organs in regulating zinc homeostasis. Upon removal of the tissues, the mice were killed by cervical dislocation. The bones were stored at -70°C until determination of zinc concentration. Upon removal, the liver was rinsed with cold phosphate buffered saline (PBS, pH 7.4). A section of the liver (~ 2 mm) was sliced off from the largest lobe of the liver and was immediately submerged in chilled acetone for assessment of histochemically reactive zinc. After overnight at -70 °C, the acetone-treated liver slice was transferred to a base mold (15 x 15 x 5 mm; Fisher Scientific, Ottawa, Ontario) that was pre-filled with O.C.T Compound 43 Tissue-Tek (OCT; Sakura Finetek, Torrance, CA). The liver slice was left at room temperature for approximately 2 min to allow the liver slice to sink into OCT. The liver slice embedded in OCT was then kept at - 20°C until cryostat sectioning. After slicing off the liver section for histochemically reactive zinc analysis, the remaining liver was quickly minced up with scissors and divided into two portions: one portion for total RNA extraction and the other portion for 109Cd-hemoglobin affinity assay. Both portions were separately frozen in liquid nitrogen immediately and stored at -70°C until analyses. The small intestine (-19-23 cm) was removed and rinsed in cold PBS (pH 7.4). An approximately 3-mm-long section was cut from the ileum for the assessment of histochemically reactive zinc using the same tissue preservation protocol described above for the liver. In mice, the ileum has a diameter of 3 - 5 mm, which is distinctly smaller than the cecum (~ 10 mm; Cook, 1965). The remaining small intestine, including duodenum, jejunum, and ileum, was quickly minced, mixed and divided into two portions. One portion of the minced small intestine was used for total RNA extraction and the other portion was used for 109Cd-hemoglobin affinity assay. Both portions were separately frozen in liquid nitrogen immediately and stored at -70°C until analyses. Assessment of body zinc status Body zinc status was assessed by measuring the femur and tibia zinc concentrations. The muscle tissue attached to the bones was removed completely. Prior to performing the procedure, all crucibles and glassware were acid-washed by soaking them in a nitric acid bath (2 N) for 48 h, followed by rinsing with double-deionized water thoroughly to prevent potential contamination. 44 The bones were wet-ashed using the established protocol (Clegg et al, 1981). Briefly, the acid-washed crucibles were first dried at 100°C for 4 h and then cooled in a desiccator. The weight of the cooled crucible was obtained and recorded. The bones were transferred into the cooled crucibles and weighed to obtain the wet weight of the bones. After drying at 100°C overnight, the dried bones, together with the crucible, were cooled in a desiccator and weighed to obtain the dry weight of the bones. The dried bones were transferred to acid-washed Kimax glass test tubes (16 x 120 mm; Fisher Scientific, Ottawa, Ontario) and digested in concentrated nitric acid (16 N; 1.5 ml) at 100°C for 5 h. The test tubes were covered with acid-washed glass marbles to prevent evaporation. Once the acid digestion was completed, the digested bone samples were transferred quantitatively into 5-ml acid-washed volumetric flasks and brought to the volume with double-deionized water, followed by transferring the digested sample (5 ml) to scintillation vials (VWR, Mississauga, Ontario). Finally, the samples were diluted with double-deionized water to appropriate concentration for the determination of zinc concentration using flame atomic absorption spectroscopy (Atomic Absorption Spectrophotometer, model 2380, Perkin Elmer, Norwalk, CT, USA). The dilution factor was determined based on preliminary testing. Bone zinc concentrations were normalized on the basis of dried bone weight. Assessment of metallothionein concentrations Metallothionein concentrations were determined using a modified ^cadmium-hemoglobin affinity assay (Eaton and Cherian, 1991). Sample preparation: Frozen liver (~ 0.4 g) and small intestine (~ 0.5 g) samples were weighed and put into plastic test tubes (12 x 75 mm, VWR culture disposable plastic tubes; 45 VWR, Mississauga, Ontario.) containing 500 u l of cold Tris-HCl buffer (10 mM, pH 7.4) on ice. The tissues were homogenized with Polytron (PCU-2110, Brinkmann Instrument, Rexdale, Ontario) for 20 - 30 sec. After homogenization, the polytron probe was rinsed with another 500 ul of cold Tris-HCl buffer (10 mM, pH 7.4), which brought the final volume to 1 ml. The homogenized samples were kept on ice before centrifugation (1,500 rpm, 10 min, 4°C; IEC Centra-7R refrigerated centrifuge, E C Electronics Corporation, NY, USA). After centrifugation, a portion of the supernatant (200 ul) was transferred into a microcentrifuge tube and kept at -70°C before determination of protein content. Another portion of the supernatant (800 ul) was transferred into another microcentrifuge tube followed by heating the sample in a boiling water bath for 2 min. The denatured samples were then quickly cooled on ice before centrifugation (14,000 g, 5 min, 4°C; Eppendorf Microcentrifuge 5415D, Westbury, NY, U.S.A.). The supernatant was then transferred into a new microcentrifuge tube and was kept at -70°C until the determination of M T concentration. Solution preparation: 1 0 9 C d in HCI (3.7 MBq/ug Cd; 100 uCi/ug Cd) was purchased from Amersham Biosciences (Quebec, Canada) The 1 0 9 C d in HCI was used to prepare 1 0 9 C d stock solution [100 uCi in 250 ul of Tris-HCl buffer (10 mM; pH 7.4)]. To prepare 1 0 9 C d working solution (0.4 ug CdCl 2/ml; 0.2 uCi 1 0 9Cd/ml), 1.6 ml of the cold CdCl 2 solution [5 ug CdCl 2/ml Tris-HCl buffer (30 mM; pH 8.0)] was diluted with 18.4 ml of Tris-HCl buffer (30 mM, pH 8.0) followed by adding 10 ul of 1 0 9 C d stock solution. The hemoglobin solution (2%; w/v) was prepared by dissolving 2 g hemoglobin (Sigma-Aldrich, Oakville, Ontario) in 100 ml Tris-HCl (30 mM; pH 8.0). mCd-hemoglobin affinity assay: To carry out the assay, 200 ul of heat-denatured supernatant, 100 u l of Tris-HCl buffer (30 mM; pH 8.0) and 100 ul of 1 0 9 C d working solution 46 were mixed well in a microcentrifuge tube. The reaction mixture was then incubated for 15 min at room temperature. At the end of the incubation period, 100 ul of hemoglobin solution was added into each reaction tube followed by boiling (2 min), chilling on ice (4 min), and centrifugation (14,000 g; 10 min) to remove unbound 1 0 9 C d . This hemoglobin-washing step was repeated two more times. After the final hemoglobin-washing, the supernatant (400 ul) was carefully transferred to a Falcon polypropylene disposable test tube (12 x 75 mm, VWR, Mississauga, Ontario) and was kept at 4°C before determining the radioactivity in each sample using a gamma counter (1277 Gammamaster, L K B Wallac, Turku, Finland). The samples were kept at 4°C for no more than 24 hours prior to the determination of the radioactivity. Calculation: the following formula was used to calculate M T concentration: nmole Cd bound/ml = (Ctss - Cts BKo) x (1.78/CtsT) x DF Ctss = sample counts CtssKG = background counts [300 ul Tris-HCl buffer (30 mM; pH 8.0) + 100 (a.1 1 0 9 C d working solution + 300 ul hemoglobin solution] CtsT = total counts [300 ul Tris-HCl buffer (30 mM; pH 8.0) + 100 ul 1 0 9 C d working solution] DF = dilution factor 1.78 = Cd in each sample (0.04 ug)/[atomic weight of Cd (0.1124 ug/nmol) x M T sample size (0.2 ml)] 47 Assessment of the abundance of histochemically reactive zinc Zinquin ethyl ester (VWR C A N L A B , Mississauga, Ontario) was dissolved in 100% dimethyl sulfoxide (DMSO; Sigma-Aldrich, Oakville, Ontario) to make a stock solution (5 mM). Zinquin working solution (50 uM) was prepared by diluting the stock solution with PBS (pH 7.4). Zinquin working solution was kept at -20°C for no more than 100 days prior to use. To assess the abundance of the histochemically reactive zinc, frozen liver and small intestine samples embedded in OCT were cryosectioned (5 p.m in thickness), mounted on glass slides and kept at - 20°C until analysis. The slides were first washed by covering the entire slide with PBS (pH 7.4) and gently swirling the PBS around for approximately three seconds. The washing step was repeated three times to remove residual OCT followed by tilting the slides to remove residual PBS. To stain histochemically reactive zinc, the entire tissue was covered with the Zinquin working solution (- 35 - 50 ul) and left at room temperature for 20 min. After staining, the tissue slides were gently rinsed with PBS (pH 7.4) three times to remove excess Zinquin. The slides were then covered with glass cover slides. Zinquin-dependent fluorescence image was then visualized using a fluorescence microscope (Carl Zeiss Vision, Axiovision 4.1, Oberkochen, Germany) at 100 X and 200 X magnifications. The fluorescent light was passed through an emission filter with a cut off wavelength of 420 nm (Carl Zeiss, Oberkochen, Germany). To maintain the consistency of the images, all the images were photographed with a digital camera (AxioCam M R M , Carl Zeiss, Oberkochen, Germany) using the same exposure time (40 ms for the liver; 25 ms for the small intestine). Fifteen percent of light was used for both tissues. The relative 48 brightness of the images was evaluated independently by eight volunteers blinded to treatment. Assessment of mRNA levels of zinc transporters Total RNA isolation: The liver and small intestine samples (~ 0.08 - 0.12g) were homogenized in 1 ml of TRIZOL (Invitrogen, Burlington, Ontario) with a glass-Teflon tissue grinder (Fisher Scientific, Ottawa, Ontario) to isolate total RNAs according to the manufacturer's instructions. The total RNA pellets were resuspended in 100 to 200 u l of the DEPC-treated water and stored at -70°C before analyses. RNA concentrations were determined using OD (optical density) at 260 nm, which was determined using a microplate reader (SpectraMax Plus 384, Molecular Devices, Sunnyvale, C A , USA). Reverse Transcription-Polymerase Chain Reaction (RT-PCR): RT-PCR was performed using the ThermoScript RT-PCR System plus Platinum Taq DNA Polymerase (Invitrogen, Burlington, Ontario) according to the manufacture's instruction. Briefly, cDNAs were obtained from total RNAs (2 ug) using reverse transcription (GeneAmp PCR System 2400; Perkin Elmer). The resulting cDNA (lul, which was equivalent to 0.1 pg of total RNAs) was then amplified with thermocycler (Eppendorf Mastercycler Gradient, Brinkmann, Mississauga, Ontario). The same cDNA preparation technique was used for all the samples. The specific forward and reverse primers used to generate the product fragments were as follows: ZnT-1: 5'- T G A A G G C G G A C C A G G C A G A G 3 ' and 5'-A G A C A T G T A G C T C A T G G A C T T C - 3 ' (519 bp; Genbank accession code BC052166); ZnT-2: 5 ' -AGCCATTGCCCAGAATGCTG-3' and 5 ' -CATGGATCTTGTTCAATTTTTGG-3' (473 bp; Genbank accession code AK031425); ZnT-4: 5'-49 T T G C A G T T A A T G T A A T A A T G G G G T T - 3 * and 5 * - G A C A A T T T G C A C A A G T T C - T G A T C -3* (590 bp; Genbank accession code AF003747); Nramp2: 5'-ATGCTG A C T C A G -TGGTGTCC-3' and 5' -GAACTTACTGGCTGGCTAGC-3' (739 bp; Wardrop and Richardson, 1999); p-actin: 5 ' -TATGGAGAAGATTTGGCACC-3' and 5'-C C A C C A A T C C A C A C A G A G T A - 3 ' (786 bp; Lee et al., 2003). After a hot start (95°C; 2 min), the samples were amplified at 95°C/30 sec, 60°C/30 sec, 72°C/1 min with a single final extension at 72°C/6 min for ZnT-1 and -4, and at 95°C/30 sec, 57°C/30 sec, 72°C/1 min with a single final extension at 72°C/7 min for ZnT-2, Nramp2 and P-actin. For each transporter, preliminary experiments were conducted to determine the optimum number of cycles. The number of cycles was chosen to ensure that the amplification was within the linear phase of the amplification curve and that the amplification did not yield nonspecific PCR products. The number of cycles used were 30, 30, 28, and 28 for ZnT-1, and -4, Nramp2, and P-actin, respectively, for both the liver and small intestine. The number of cycles for ZnT-2 was 33 and 30 for the liver and small intestine, respectively. Two negative controls, the cDNA blank and the primer blank, were included for each targeted gene to ensure the specificity of RT-PCR. The PCR products were eluted on agarose gel (1.5 %) and the optical density of the bands was quantified using Kodak ID Image Analysis Software (version 3.6; Eastman Kodak Company, Rochester, NY). The steady state mRNA levels of ZnT-1, -2, and -4, and Nramp2 were normalized on the optical density of the corresponding P-actin. Statistical analyses The differences among zinc treatment and treatment duration means in MT-WT mice were analyzed using two-way A N O V A . Significant main effects were further analyzed by 50 LSMeans Differences Student's t test (JMP Release 5.0). The differences between M T - W T and M T - K O mice were analyzed using two-way ANOVA. Significant main effects were further analyzed by LSMeans Difference Student's t test (JMP release 5.0). Selected comparisons with baseline values were analyzed using Student's t test (JMP release 5.0). R E S U L T S Body Zinc Status Body weight gain. In MT-WT mice, body weight gain was not affected by dietary zinc intake or treatment duration (Table II-1). No interaction effect on body weight gain was found between zinc treatment and treatment duration. In M T - K O mice, high-zinc M T - K O mice had a significant reduction in body weight gain compared to adequate-zinc M T - K O mice, demonstrating intolerance of M T - K O mice to high-zinc intake (77%; p < 0.05; Table II-1). However, low-zinc M T - K O mice had similar body weight gain as adequate-zinc M T -K O mice. Comparing M T - K O mice to M T - W T mice, M T - K O mice had an overall lower body weight gain (p < 0.05). Overall zinc treatment effect was close but not yet reached statistical difference (p < 0.05). No interaction effect on body weight gain was observed between strain and zinc treatment. However, at high-zinc intake, high-zinc M T - K O mice had an 80% reduction in body weight gain compared to high-zinc M T - W T mice (p < 0.05). Feed intake. In M T - W T mice, feed intake was mainly affected by treatment duration (p < 0.05) but not by zinc treatment (p < 0.05). No interaction effect on feed intake was observed between zinc treatment and treatment duration in MT-WT mice. In M T - W T mice, feed intake increased gradually from day 2, 4, 7 to day 21 regardless of zinc treatment (Table II-1). In M T - K O mice, feed intake was also unaffected by zinc treatment (Table II-1). When 51 comparing M T - K O mice to MT-WT mice, M T - K O mice had an overall lower feed intake (p < 0.05). Overall zinc treatment had no effect on feed intake. No interaction effect on feed intake was observed between strain and zinc treatment. Further analyses on the effect of treatment duration found that after 4 days of treatment, M T - K O mice had similar feed intake as MT-WT mice at low-zinc intake. At adequate- or high-zinc intake, however, feed intake in M T - K O mice reduced by 60% (p < 0.05) and 57% (p < 0.05), respectively compared to that in MT-WT mice. At high-zinc intake, M T - K O mice showed signs of weakness that was probably secondary to zinc toxicity symptoms, and 3 M T - K O mice (8 mice in total) died after 48 hr of high-zinc intake. Bone zinc concentration. Bone zinc was assessed by the femur and tibia zinc concentration combined (Figure II-1). In MT-WT mice, bone zinc concentration was overall affected by zinc treatment and treatment duration (p's < 0.05). Overall, bone zinc concentration increased as zinc intake increased. Interaction effect on bone zinc concentration was observed between zinc treatment and treatment duration (p < 0.05). Further analyses found that bone zinc concentration was not affected by low-zinc intake after 2 days of treatment; however, compared to that at adequate-zinc intake, bone zinc concentration at low-zinc intake significantly decreased after 4 days of treatment (35%; p < 0.05) and remained at the reduced level for the rest of the feeding trial. At adequate-zinc intake, bone zinc concentration was unaffected by treatment duration. At high-zinc intake, bone zinc concentration increased significantly (44%) after 2 days of treatment compared to that at adequate-zinc intake and remained elevated 4 (32%), 7 (76%), and 21 (43%) days after treatment O's < 0.05). 52 In M T - K O mice, overall zinc treatment had no effect on bone zinc concentration. Further analyses found that although bone zinc concentration was not affected by low- or high-zinc intake compared to that at adequate-zinc intake, bone zinc concentration in high-zinc M T - K O mice was significantly higher than that in low-zinc M T - K O mice (41%; p < 0.05; Figure II-1). MT-WT vs. MT-KO mice. Prior to zinc treatment, bone zinc concentration in M T - K O mice was significantly higher than that in MT-WT mice (44%; p < 0.05; Figure II-1). When comparing MT-WT mice to M T - K O mice, M T - K O mice had an overall higher bone zinc concentration than MT-WT mice (p < 0.05). Overall bone zinc concentration was also affected by zinc treatment (p < 0.05). Higher bone zinc concentration was found in high-zinc mice. No interaction effect on bone zinc concentration was observed between strain and zinc treatment. Metallothionein concentration Liver MT-WT mice. Prior to zinc treatment, twenty-three-day old weanling MT-WT mice had an average of 11.5 ug MT/g liver (Figure II-2). In MT-WT mice, hepatic M T concentration was affected by treatment duration and zinc treatment (p's < 0.05). Overall, hepatic M T concentration increased as zinc treatment increased. Hepatic M T concentration was the lowest after 21 days of treatment. Interaction effect on hepatic M T concentration was observed between zinc treatment and treatment duration (p < 0.05). Compared to baseline M T concentration, hepatic M T concentrations were higher after 2 days of treatment in all treatment groups (p < 0.05). Post-hoc analyses showed that after 2 days of treatment, 53 hepatic M T concentration at low-zinc intake was similar to that at adequate- zinc intake; however, hepatic M T concentration was significantly higher at high-zinc intake (49%), indicating a hepatic M T induction by high-zinc intake. After 4 or 7 days of treatment, hepatic M T concentration in low-zinc group was significantly lower than that in adequate-zinc group by 86% and 70%, respectively, while high-zinc MT-WT mice had 121% and 70% higher hepatic M T concentration, respectively, than adequate MT-WT mice (p's < 0.05). Adequate-zinc MT-WT mice maintained a relatively stable hepatic M T concentration throughout the feeding trial. After 21 days of treatment, zinc treatment no longer had effect on hepatic M T concentration, and hepatic M T concentrations were generally the lowest in all treatment groups. Small Intestine MT-WT mice. Prior to treatment, twenty-three-day old weanling MT-WT mice had an average of 0.24 ug MT/g of small intestine (Figure II-3). In MT-WT mice, small intestinal M T concentration was affected by zinc treatment and treatment duration (p's < 0.05). Interaction effect on small intestinal M T concentration was observed between zinc treatment and treatment duration (p < 0.05). Overall, small intestinal M T concentration increased as zinc treatment increased. Small intestinal M T concentration seemed to peak after 7 days of treatment. Further analyses found that compared to adequate-zinc MT-WT mice, low-zinc M T - W T mice had significantly lower small intestinal M T concentration after 4 (85%) or 7 days (60%) of treatment (p's < 0.05) but not after 2 or 21 days of treatment. On the other hand, high-zinc MT-WT mice had significantly higher small intestinal M T concentration compared to adequate-zinc M T - W T mice regardless of treatment duration. At 54 low-zinc intake, small intestinal M T concentration increased by 150% from baseline after 2 days of treatment and remained similar regardless of treatment duration (p < 0.05). At adequate-zinc intake, small intestinal M T concentration increased by 16-fold from baseline after 2 days of treatment and remained at the same concentration until the end of the feeding trial. At high-zinc intake, small intestinal M T concentration by 92-fold from baseline after 2 days of treatment, followed by a further increase of 43% after 4 days of treatment (p < 0.05). The increase in small intestinal M T concentration in response to high-zinc intake continued and peaked after 7 days of treatment. MT-WT mice with 7 days of high-zinc intake had the highest small intestinal M T concentration than other MT-WT mice. After 21 days of treatment, however, small intestinal M T concentration decreased by 67 % from that on day 7 and was 24% lower than that on day 2. The abundance of histochemically reactive zinc Liver MT-WT mice. The images selected were the representative pictures for each group. No dramatic discrepancies in brightness were observed among the replications (n = 6; 2 slices/mouse) in each group. The abundance of hepatic histochemically reactive zinc in M T -WT mice appeared to be unaffected by zinc treatment or treatment duration as indicated by the similar overall brightness (Figure II-4). The image on day 7 at high-zinc intake, however, was brighter and showed scattered bright spots compared to those on day 7 at low- or adequate-zinc intake, indicating higher hepatic histochemically reactive zinc abundance after 7 days of high-zinc intake. 55 MT-KO mice. Compared to adequate-zinc M T - K O mice, low-zinc M T - K O mice appeared to have similar abundance of hepatic histochemically reactive zinc after 4 days of treatment, as the images shared similar overall brightness (Figure II-4). However, the image of high-zinc M T - K O mice after 4 days treatment was brighter and showed scattered bright spots compared to that of adequate-zinc M T - K O mice MT-WT mice vs. MT-KO mice. The basal hepatic histochemically reactive zinc was more abundant in M T - W T mice than that in M T - K O mice (Figure II-4). After 4 days of treatment, hepatic histochemically reactive zinc at low- or adequate-zinc intake appeared to be more abundant in MT-WT mice than that in M T - K O mice. At high-zinc intake, however, hepatic histochemically reactive was more abundant in M T - K O mice than that in M T - W T mice. Small intestine MT-WT mice. Compared to adequate-zinc MT-WT mice, low-zinc MT-WT mice were less abundant in small intestinal histochemically reactive zinc regardless of treatment duration, indicating that low-zinc intake reduced the abundance of small intestinal histochemically reactive zinc (Figure II-5). However, treatment duration did not appear to have affected the abundance of small intestinal histochemically reactive zinc of low-zinc M T - W T mice as indicated by the similar brightness. On the other hand, high-zinc M T - W T mice were more abundant in small intestinal histochemically reactive zinc, especially in the circular and longitudinal muscular layers, compared to adequate-zinc MT-WT mice, indicating that zinc supplementation increased the abundance of small intestinal histochemically reactive zinc. In adequate-zinc mice, the abundance of small intestinal 56 histochemically reactive zinc appeared to be unaffected after 2 or 4 days of treatment, but it was more abundant after 7 days of treatment. On day 21, small intestinal histochemically reactive zinc was much less abundant compared to that on day 7. In high-zinc mice, small intestinal histochemically reactive zinc was more abundant, especially in the circular and longitudinal muscular layers, after 4 or 7 days of treatment. Small intestinal histochemically reactive zinc on day 21 was not as abundant as those on day 4 and 7 but shared a similar abundance as that on day 2, regardless of zinc treatment. MT-KO mice. Compared to adequate-zinc M T - K O mice, low-zinc M T - K O mice were less abundant in small intestinal histochemically reactive zinc especially in the circular muscular layer, indicating that low-zinc intake reduced the abundance of histochemically reactive zinc in M T - K O mice (Figure II-5). On the other hand, high-zinc M T - K O mice were more abundant in small intestinal histochemically reactive zinc compared to adequate-zinc M T - K O mice. MT-WT mice vs. MT-KO mice. The basal small intestinal histochemically reactive zinc in MT-WT mice was more abundant than that in M T - K O mice (Figure II-5). After 4 days of low- or adequate-zinc intake, MT-WT mice continued to have higher abundance in small intestinal histochemically reactive zinc than M T - K O mice. At high-zinc intake, M T -WT and M T - K O mice shared similar overall brightness; however, M T - W T mice had especially higher small intestinal histochemically reactive zinc abundance in the circular and longitudinal muscular layers than M T - K O mice. 57 The abundance of ZnT-1 mRNA Liver MT-WT mice. Prior to zinc treatment, the twenty-three-day old M T - W T mice had an average hepatic ZnT-1 mRNA abundance of 1. In MT-WT mice, neither zinc treatment nor treatment duration had an effect on the abundance of hepatic ZnT-1 mRNA (Figure II-6). However, interaction effect on the abundance of hepatic ZnT-1 mRNA was observed between zinc treatment and treatment duration (p < 0.05). Overall, hepatic ZnT-1 mRNA level increased as zinc treatment increased on day 2. However, hepatic ZnT-1 mRNA level decreased as zinc treatment increased after 21 days of treatment. Further analyses found that after 2 days of treatment, high-zinc M T - W T mice had 25% higher hepatic ZnT-1 mRNA level compared to low-zinc M T - W T mice, while after 21 days of treatment, high-zinc M T -WT mice had 29% lower hepatic ZnT-1 mRNA level compared to low-zinc M T - W T mice (p <0.05). MT-KO mice. Prior to zinc treatment, twenty-three-day old M T - K O mice had an average ZnT-1 mRNA abundance of 1.2. After 4 days of treatment, low-zinc mice had significantly lower (27%; p < 0.05) hepatic ZnT-1 mRNA level compared to adequate-zinc mice (Figure II-6). On the other hand, high zinc-intake had no effect on the level of hepatic ZnT-1 mRNA compared to adequate-zinc intake in M T - K O mice. MT-WT mice vs. MT-KO mice. Prior to zinc treatment, no difference in hepatic ZnT-1 mRNA level was found between twenty-three-day old MT-WT and M T - K O mice (Figure II-6). Overall, the abundance of hepatic ZnT-1 mRNA was not affected by strain or zinc treatment. No interaction effect on the abundance of hepatic ZnT-1 mRNA was observed between strain and zinc treatment, although it was close to being significant (p = 0.06). 58 Small intestine MT-WT mice. Prior to zinc treatment, twenty-three-day old M T - W T mice had an average small intestinal ZnT-1 mRNA abundance of 0.6. In M T - W T mice, neither zinc treatment nor treatment duration had an effect on the abundance of small intestinal ZnT-1 mRNA (Figure II-7). No interaction effect on the abundance of small intestinal ZnT-1 mRNA was observed between zinc treatment and treatment duration. MT-KO mice. Prior to zinc treatment, twenty-three-day old M T - K O mice had an average small intestinal ZnT-1 mRNA abundance of 1.3 (Figure II-7). After 4 days of treatment, low-zinc mice had significantly higher small intestinal ZnT-1 mRNA level (43%; p < 0.05) compared to adequate-zinc mice. On the other hand, high-zinc mice had similar small intestinal ZnT-1 mRNA level as adequate-zinc mice after 4 days of treatment. MT-WT mice vs. MT-KO mice. Prior to zinc treatment, twenty-three-day old M T -WT mice had 54% lower small intestinal ZnT-1 mRNA level compared to M T - K O mice (p = 0.0006; Figure II-7). The abundance of small intestinal ZnT-1 mRNA was not affected by strain or zinc treatment. No interaction effect on the abundance of small intestinal ZnT-1 mRNA level was observed between strain and zinc treatment, although it was close to being significant (p = 0.056). The abundance of ZnT-2 mRNA Liver MT-WT mice. Prior to zinc treatment, twenty-three-day old MT-WT mice had an average hepatic ZnT-2 mRNA abundance of 1.0. In M T - W T mice, neither zinc treatment nor treatment duration affected the abundance of hepatic ZnT-2 mRNA (Figure II-8). No 59 interaction effect on the abundance of hepatic ZnT-2 mRNA was found between zinc treatment and treatment duration. MT-KO mice. Prior to zinc treatment, twenty-three-day old M T - K O mice had an average hepatic ZnT-2 mRNA abundance of 0.9 (Figure II-8). After 4 days of treatment, low-zinc M T - K O mice had significantly lower hepatic ZnT-2 rnRNA level compared to adequate-zinc mice (40%; p < 0.05). High-zinc mice, on the other hand, had similar hepatic ZnT-2 mRNA level as adequate-zinc mice. MT-WT mice vs. MT-KO mice. Prior to zinc treatment, no difference in hepatic ZnT-2 mRNA level was observed between twenty-three-day old M T - W T and M T - K O mice (Figure II-8). The abundance of hepatic ZnT-1 mRNA was not affected by strain; however, it was affected by zinc treatment (p < 0.05). Overall, the abundance of small intestinal ZnT-1 mRNA level was lower in low-zinc mice than that in adequate-zinc mice. No interaction effect on the abundance of small intestinal ZnT-1 mRNA level was observed between strain and zinc treatment. Further analyses on zinc treatment found that in both MT-WT and M T -K O mice, low-zinc mice had significantly lower small intestinal ZnT-1 mRNA level compared to the respective adequate-zinc mice (MT-WT mice: 57%; M T - K O mice: 43%; p's <0.05) . Small intestine MT-WT mice. Prior to zinc treatment, twenty-three-day old M T - W T mice had an average small intestinal ZnT-2 mRNA abundance of 0.9 (Figure II-9). The abundance of small intestinal ZnT-2 mRNA was affected by treatment duration (p < 0.05) but not zinc treatment. Overall, small intestinal ZnT-2 mRNA level was higher after 21 days of treatment. 60 Interaction effect on the abundance of small intestinal ZnT-2 mRNA was observed between zinc treatment and treatment duration (p < 0.05). Further analyses found that low-zinc M T -WT mice had lower small intestinal ZnT-2 mRNA level compared to adequate-zinc mice after 4 days of treatment (36%; p < 0.05). However, low-zinc MT-WT mice had higher small intestinal ZnT-2 mRNA level compared to adequate-zinc mice after 21 days of treatment (31%; p < 0.05). MT-KO mice. Prior to zinc treatment, twenty-three-day old M T - K O mice had an average small intestinal ZnT-2 mRNA abundance of 1.2 (Figure II-9). After 4 days of treatment, the abundance of small intestinal ZnT-2 mRNA in M T - K O mice was not affected by low-, adequate- or high-zinc intake. MT-WT mice vs. MT-KO mice. Prior to zinc treatment, no difference in small intestinal ZnT-2 mRNA level was observed between twenty-three-day old MT-WT and M T -K O mice (Figure II-9). The abundance of small intestinal ZnT-2 mRNA was affected by strain (p < 0.05) but not zinc treatment. Overall, MT-WT mice had higher small intestinal ZnT-2 mRNA levels than M T - K O mice. No interaction effect on the abundance of small intestinal ZnT-2 mRNA was observed between strain and zinc treatment, although it was close to being significant (p = 0.07). Further analyses found that after 4 days of treatment, adequate-zinc MT-WT mice had significantly higher small intestinal ZnT-2 mRNA level compared to adequate-zinc M T - K O mice (100%; p < 0.05); high-zinc MT-WT mice also had significantly higher small intestinal ZnT-2 mRNA level compared to high-zinc M T - K O mice (44%; p < 0.05). 61 Abundance of ZnT-4 mRNA Liver MT-WT mice. Prior to zinc treatment, twenty-three-day old M T - W T mice had an average hepatic ZnT-4 mRNA abundance of 0.8 (Figure 11-10). The abundance of hepatic ZnT-4 mRNA was affected by zinc treatment (p < 0.05) and treatment duration (p < 0.05). Interaction effect on the abundance of hepatic ZnT-4 mRNA was observed between zinc treatment and treatment duration (p < 0.05). Overall, hepatic ZnT-4 mRNA level was lower after 2 or 7 days of treatment compared to that after 4 or 21 days of treatment, and adequate-zinc MT-WT mice had lower hepatic ZnT-4 mRNA levels compared to low- or high-zinc mice after 2 or 7 days of treatment. Post-hoc analyses found that after 2 days of treatment, high-zinc M T - W T mice had higher hepatic ZnT-4 mRNA level by 132% (p < 0.05) compared to adequate-zinc MT-WT mice, while low-zinc MT-WT mice had similar hepatic ZnT-4 mRNA level compared to adequate-zinc M T - W T mice. After 7 days of treatment, low- (75%) or high-zinc (58%) MT-WT mice had significantly higher hepatic ZnT-4 mRNA level compared to adequate-zinc M T - W T mice (p's < 0.05). At low-zinc intake, hepatic ZnT-4 mRNA level significantly decreased by 50% from baseline after 2 days of treatment, followed by a 150% increase after 4 of treatment (p < 0.05). As treatment duration increased, hepatic ZnT-4 mRNA level decreased by 30% and reached baseline value after 7 days of treatment (p < 0.05). Hepatic ZnT-4 mRNA level in low-zinc MT-WT mice maintained at baseline value after 21 days of treatment. At adequate-zinc intake, hepatic ZnT-4 mRNA level decreased by 80% from baseline after 2 days of treatment, followed by a significant increase of 400% after 4 days of treatment (p's < 0.05). After 7 days of treatment, hepatic ZnT-4 mRNA level decreased by 60% from day 4, while 62 after 21 days of treatment, hepatic ZnT-4 mRNA level significantly increased by 125% from day 7 and was back to baseline value (/?'s < 0.05). At high-zinc intake, hepatic ZnT-4 mRNA level significantly decreased by 38% from baseline after 2 days of treatment, followed by a 120% increase after 4 days of treatment (p's < 0.05). After 7 days of treatment, hepatic ZnT-4 mRNA level significantly decreased by 36% from day 4 (p < 0.05). After 21 days of treatment, hepatic ZnT-4 mRNA in high-zinc MT-WT mice remained unchanged from that on day 7. Overall, there was a significant decrease in hepatic ZnT-4 mRNA level from baseline after 2 days of treatment regardless of zinc intake, followed by a significant increase occurred between 2 and 4 days after treatment. MT-KO mice. Prior to zinc treatment, twenty-three-day old M T - K O mice had an average hepatic ZnT-4 mRNA abundance of 1.0 (Figure 11-10). After 4 days of treatment, zinc treatment had no effect on the abundance of hepatic ZnT-4 mRNA in M T - K O mice. However, hepatic ZnT-4 mRNA levels in low- (40%), adequate-(30%), and high-zinc (30%) mice were lower than that at baseline (p's < 0.05). MT-WT mice vs. MT-KO mice. Prior to zinc treatment, no difference in hepatic ZnT-4 mRNA level was observed between twenty-three-day old MT-WT and M T - K O mice (Figure 11-10). The abundance of hepatic ZnT-4 mRNA was affected by strain (p < 0.05) but not zinc treatment. No interaction effect on the abundance of hepatic ZnT-4 mRNA was observed between strain and zinc treatment. Overall, MT-WT mice had higher hepatic ZnT-4 mRNA level than M T - K O mice. Further analyses on strain differences found that the differences were observed in all three zinc treatment groups, as hepatic ZnT-4 mRNA levels in M T - K O mice were significantly lower than those in M T - W T mice after 4 days of treatment (low-zinc: 67%; adequate-zinc: 27%; high-zinc: 47%; p's < 0.05). 63 Small intestine MT-WT mice. Prior to zinc treatment, twenty-three-day old M T - W T mice had an average small intestinal ZnT-4 mRNA abundance of 1.0 (Figure 11-11). The abundance of small intestinal ZnT-4 mRNA was affected by treatment duration (p < 0.05) but not zinc treatment. No interaction effect on the abundance of small intestinal ZnT-4 mRNA was observed between zinc treatment and treatment duration. Overall, small intestinal ZnT-4 mRNA level was lower after 2 days of treatment compared to those on other time points. MT-KO mice. Prior to zinc treatment, twenty-three-day old M T - K O mice had an average small intestinal ZnT-4 mRNA abundance of 1 (Figure II-11). Although after 4 days of treatment, the abundance of small intestinal ZnT-4 mRNA was unaffected by zinc treatment, significant decrease in small intestinal ZnT-4 mRNA level from baseline was observed after 4 days of low- (27%), adequate-(36%), or high-zinc (36%) intake (p's < 0.05). MT-WT mice vs. MT-KO mice. Prior to zinc treatment, no difference in ZnT-4 mRNA level was observed between twenty-three-day old MT-WT and M T - K O mice (Figure 11-11). The abundance of small intestinal ZnT-4 mRNA was affected by strain (p < 0.05) but not zinc treatment. Interaction effect on the abundance of small intestinal ZnT-4 mRNA level was observed between strain and zinc treatment (p < 0.05). Overall, MT-WT mice had higher small intestinal ZnT-4 mRNA level than M T - K O mice. Further analyses found that small intestinal ZnT-4 mRNA level was significantly lower (40%; p < 0.05) in adequate-zinc M T - K O mice compared to adequate-zinc MT-WT mice. 64 Abundance of Nramp2 mRNA Liver MT-WT mice. Prior to zinc treatment, twenty-three-day old MT-WT mice had an average hepatic Nramp2 mRNA abundance of 0.6 (Figure 11-12). The abundance of hepatic Nramp2 mRNA was affected by duration (p < 0.05) but not zinc treatment. No interaction effect on the abundance of Nramp2 mRNA was observed between zinc treatment and treatment duration. Overall, hepatic ZnT-4 mRNA level was lower after 7 or 21 days of treatment than those after 2 or 4 days of treatment. MT-KO mice. Prior to zinc treatment, twenty-three-day old M T - K O mice had an average hepatic Nramp2 mRNA abundance of 0.8 (Figure 11-12). After 4 days of treatment, hepatic Nramp2 mRNA level in low-zinc mice remained similar to baseline value, while those in adequate- or high-zinc mice increased significantly (100% and 80%; p's < 0.05). Furthermore, adequate-zinc mice had significantly higher hepatic Nramp2 mRNA level compared to low- or high-zinc mice after 4 days of treatment (100% and 45%, respectively; p's<0.05). MT-WT mice vs. MT-KO mice. Prior to zinc treatment, no difference in Nramp2 mRNA level was observed between twenty-three-day old MT-WT and M T - K O mice (Figure 11-12). The abundance of hepatic Nramp2 mRNA was affected by zinc treatment (p < 0.05) but not strain (p = 0.056). No interaction effect on the abundance of hepatic Nramp2 mRNA was observed between strain and zinc treatment. Overall, adequate-zinc mice had higher hepatic Nramp2 mRNA level than low- or high-zinc mice. Further analyses found that after 4 days of adequate-zinc intake, M T - K O mice had significantly higher hepatic Nramp2 65 r r i R N A than MT-WT mice (45%; p < 0.05). MT-WT and M T - K O mice shared similar hepatic Nramp2 mRNA level after 4 days of low- or high-zinc intake. Small intestine MT-WT mice. Prior to zinc treatment, twenty-three-day old M T - W T mice had an average small intestinal Nramp2 mRNA abundance of 0.4 (Figure 11-13). The abundance of small intestinal ZnT-4 mRNA was affected by treatment duration (p < 0.05) but not zinc treatment. No interaction effect on the abundance of small intestinal Nramp2 mRNA level was observed between zinc treatment and treatment duration. Overall, small intestinal Nramp2 mRNA level was lower after 7 days of treatment than those after 2 or 4 days of treatment. MT-KO mice. Prior to zinc treatment, twenty-three-day old M T - K O mice had an average small intestinal Nramp2 mRNA abundance of 0.7 (Figure 11-13). After 4 days of treatment, small intestinal Nramp2 mRNA level remained unchanged from baseline in low-or high-zinc mice but not adequate-zinc mice. Adequate-zinc M T - K O mice had 100% (p < 0.05) higher small intestinal Nramp2 mRNA level than baseline M T - K O mice. After 4 days treatment, adequate-zinc M T - K O mice had significantly higher small intestinal Nramp2 mRNA level compared to low- (36%) or high-zinc (36%) M T - K O mice (p's < 0.05). MT-WT mice vs. MT-KO mice. Prior to zinc treatment, no difference in the abundance of small intestinal Nramp2 mRNA was observed between twenty-three-day old M T - W T and M T - K O mice (Figure 11-14). The abundance of small intestinal Nramp2 mRNA was affected by strain and zinc treatment (p's < 0.05). No interaction effect on the abundance of small intestinal Nramp2 mRNA level was observed between strain and zinc 66 treatment. Overall, MT-WT mice had lower small intestinal Nramp2 mRNA level than M T -K O mice. Low-zinc mice had lower small intestinal Nramp2 mRNA level compared to adequate-zinc mice. Further analyses found that after 4 days of treatment, low-zinc M T - K O mice had 100% (p < 0.05) higher small intestinal mRNA level than low-zinc MT-WT mice, while adequate-zinc M T - K O mice had 75% (p < 0.05) higher small intestinal mRNA level than adequate-zinc MT-WT mice. No difference between high-zinc MT-WT and high-zinc M T - K O mice was observed after 4 days of treatment. DISCUSSION Body weight gain was affected by zinc treatment in MT-KO mice but not in MT-WT mice, while feed intake was not affected by zinc treatment in MT-KO and MT-WT mice Some of the first signs of zinc deficiency in mice include reduced feed intake, growth retardation, hair loss, and diarrhea. Weanling mice on extremely zinc-deficient diet (< 0.3 mg Zn/kg diet) have retarded growth rate and fail to survive for more than 8 weeks (Day and Skidmore, 1947). Rats fed zinc-deficient diet (< 1 mg Zn/kg diet) have severe growth retardation, as they have significantly lower (43 - 46%) body weight compared to rats fed zinc-adequate diet (30 mg Zn/kg diet; Szczurek et al., 2001). However, in this study, feeding low-zinc diet had no effect on feed intake and body weight gain in low-zinc MT-WT mice compared to adequate-zinc MT-WT mice. Moreover, no visible zinc deficiency symptoms (e.g. hair loss) were observed in low-zinc MT-WT mice. Severity of zinc deficiency is a function of dietary zinc intake and the duration of the dietary treatment. Lepage et al. (1999) reported that feeding a low-zinc diet (< 1 mg Zn/kg diet) to female C57BL/6 mice for 4 weeks results in a 15% reduction in body weight. However, feeding a 67 low-zinc diet (3 mg Zn/kg diet) to female C57BL/KsJ (BL/Ks) heterozygous lean mice for 6 weeks has no effect on body weight (Simon and Taylor, 2001). In general, the lower the zinc intake, the more zinc deficient the animals are. Therefore, in this study, the absence of reduced feed intake and body weight in response to low-zinc diet in MT-WT mice was likely due to the marginal low-zinc intake and the relatively short treatment duration. Similar to its genetic control mice, M T - K O mice fed low-zinc diet for 4 days had unaffected feed intake and body weight gain compared to adequate-zinc M T - K O mice. These observations are consistent with the results from previous studies. Philcox et al. (2000) reported that M T - K O mice fed a severe zinc-deficient diet (0.8 mg Zn/kg diet) are able to maintain their body weight for over 2 weeks and the weight loss does not become significant until 17 days on the zinc-deficient diet. In contrast to low-zinc intake, supplemented-zinc intake resulted in reduced body weight and high mortality after 4 days in M T - K O mice. It is well established that excessive zinc intake can induce zinc toxicity. In vitro, baby hamster kidney (BHK) cells have retarded growth in medium containing > 200 uM Zn, and most cells die when Zn concentration is increased to 800 u.M (Palmiter, 2004). In humans, daily zinc intake of 18.5 to 25 mg induces copper deficiency, and acute zinc intake of 1 to 2 g can cause bloody diarrhea (Festa et al., 1985; Fosmire, 1990). Metallothionein has high binding capacity and binding constant towards zinc. Each molecule of M T can bind to up to 7 zinc atoms, which gives M T a protective role during high-zinc intake (Jacob et al., 1998). In fact, induction of M T is one of the most important strategies in preventing zinc toxicity. Upon ingestion of excessive amount of zinc, M T synthesis is upregulated in the intestine to reduce zinc absorption (McMahon and Cousins, 1998). In addition, M T synthesis in the liver is also upregulated to 68 further reduce circulation zinc concentration (McMahon and Cousins, 1998). In this study, M T - K O mice lack the ability to synthesize the inducible MT-1 and -2 isofoms (Appendix II). Without MT-1 and MT-2 proteins, M T - K O mice were sensitive to zinc toxicity, and zinc intake at 150 mg Zn/kg diet was lethal in M T - K O mice. MT-WT mice on the other hand, showed no adverse effect on body weight after 21 days of high-zinc intake. Clearly, M T - K O mice are more susceptible to zinc toxicity. Bone zinc concentration is a good indicator of body zinc status Zinc concentration in tissues varies with tissue type, and different tissues might respond to zinc intake to different extents. For example, in severe zinc deficiency, zinc concentrations in hair, skin, heart, and muscle remain unaffected, while plasma and bone zinc concentrations drop significantly (Tapiero & Tew, 2003). Cousins and Lee-Ambrose (1992) showed that growing rats fed 180 mg Zn/kg diet have significantly higher bone zinc concentration than those fed 5 or 30 mg Zn/kg diet. Reis et al. (1991) also found that tibia zinc concentration decreases in response to the progressively lowered zinc in dam's milk as lactation day increases. The bone contains about one third of the total body zinc. Bones can accumulate zinc in times of excess, while a decline in bone zinc concentration may reflect a decreased zinc uptake in response to plasma zinc reduction (King et al., 2000). Similarly, results reported herein also showed that bone zinc concentration reduced at low-zinc intake and elevated at high-zinc intake. In Lepage et al.'s (1999) study, femur zinc shows a same trend as serum zinc in response to zinc intakes. As a result, bone zinc concentration has been used to assess total body zinc status. Although plasma zinc concentration is also used as an indicator for zinc status, due to plasma's responsibility to provide zinc to all tissues, it needs 69 to maintain a more constant zinc concentration. Plasma zinc concentration might therefore change more slowly in response to zinc intake (Tapiero & Tew, 2003). Therefore, comparing to plasma zinc concentration, bone zinc concentration might be a better indicator for body zinc status. Bone zinc concentration confirmed mild zinc-deficient status in low-zinc MT-WT mice In this study, bone zinc concentration was used to assess total body zinc. In M T - W T mice, starting from day 4 after treatment, bone zinc concentration was reduced at low-zinc intake. It was noticed that bone zinc concentration in low-zinc MT-WT mice did not reach significant reduction until 4 days after treatment. This delay reflects the duration needed to deplete the pre-existing body zinc reserve under the experimental conditions. The reduced bone zinc concentration at low-zinc intake showed that it was responsive to low-zinc intake. The significantly lowered bone zinc concentration and the unaffected body weight gain and feed intake confirmed the induction of marginal zinc deficiency in low-zinc M T - W T mice. Bone zinc concentration was higher but less responsive to zinc intakes in MT-KO mice Bone zinc concentration was higher in M T - K O mice at baseline and in low- or adequate-zinc M T - K O mice than their corresponding M T - W T mice. The reason as to why M T - K O mice have higher bone zinc concentration is presently not known. M T plays an important role in zinc homeostasis. In the absence of this important regulator of body zinc, such as in M T - K O mice, zinc homeostasis is likely compromised, resulting in elevated tissue zinc concentration. For example, Tran et al.'s (1998) found a much greater zinc uptake by 70 muscles and skin in M T - K O mice. It is therefore possible that excess zinc is dissipated among tissues to prevent potential zinc toxicity. In M T - K O mice, feeding low-zinc diet for 4 days did not significantly reduce bone zinc concentration compared to adequate-zinc M T - K O mice. This lack of effect on bone zinc concentration could be due to a combination of several reasons. First of all, the baseline bone zinc concentration was higher in M T - K O mice than that in MT-WT mice. As a result, it takes longer to deplete body zinc reserve prior to developing zinc deficiency. Inconsistent with this notion, M T - K O mice maintained a higher bone zinc concentration compared to MT-WT mice after 4 days of low- or adequate-zinc intake. Similarly, Tran et al. (1998) found that M T - K O mice have significantly higher zinc concentration in jejunum/ileum (45%). Secondly, development of zinc deficiency is a function of zinc intake and zinc treatment duration. In this study, the bone zinc concentration in low-zinc M T - K O mice was 18% lower than that in adequate-zinc M T - K O mice, but the difference is not statistically significant. It is possible that 4 days was not sufficiently long enough to deplete bone zinc concentration and that a significant reduction in bone zinc concentration could be reached in low-zinc M T -K O mice if the feeding trial was extended to a longer period. Metallothionein concentration reflected dietary zinc intake in the liver and small intestine Zinc is a potent stimulant for M T synthesis. The synthesis of M T is proportional to zinc intake in mice or rats at a broad range of zinc intakes (e.g. zinc sulfate in liquid form or 5 to 180 mg Zn/kg diet) and of treatment durations (e.g. several hours to 42 days; Reeves, 1995; Davies et al., 1998; Coyle et al., 1999; Szczurek et al., 2000). In this study, although 71 the zinc treatments and treatment durations are different from previous studies, the response of M T to zinc intake is consistent with the findings from previous studies: the higher the zinc intake, the higher the M T synthesis. Prior to zinc treatment, MT-WT mice had free access to regular rodent chow, which contained approximately 70 mg Zn/kg diet. Both hepatic and small intestinal M T concentrations in low-, adequate-, or high-zinc M T - W T mice were induced after zinc treatment, although the zinc concentrations in low- or adequate-zinc diet were lower than that in regular rodent chow. Similar results are observed in Dalton et al.'s (1996) study, in which the zinc-deficient diet (0.5-1.5 ug Zn/g diet) induces hepatic M T concentration in mice after 9, 18, or 27 days of treatment. A change in zinc intake therefore seems to induce M T regardless of zinc concentration. The initial increases in M T concentration from baseline after zinc treatment were probably due to the disruption of homeostasis. Regardless of zinc intake, hepatic M T and small intestinal M T were both at homeostatic conditions before zinc treatment. Metallothionein was stabilized at a concentration enough to maintain zinc homeostasis, and the metal binding sites of M T were probably saturated (Pattison and Cousins, 1986; Palmiter, 2004). Zinc treatment disrupted the already established zinc-homeostatic condition, and the synthesis of M T was stimulated as a result regardless of zinc intake. In the liver, M T concentrations returned to close to baseline value between 7 and 21 days after treatment regardless of zinc intake. Zinc intake no longer affected hepatic M T concentration after 21 days of treatment. Therefore, homeostasis was likely reached at some point between 7 and 21 days after treatment. In the small intestine, on the other hand, although M T concentrations also significantly decreased between 7 and 21 days after 72 treatment, small intestinal M T concentration still responded to zinc intake and a homeostatic condition was probably not yet reached on day 21. Our findings together with Reeves' (1995) study suggest an adaptation response of M T to zinc intake. In Reeves' (1995) study, intestinal M T concentration in rats fed a diet containing 350 mg Zn/kg diet peaked between day 3 and 14 and started to decline as treatment duration increased. While Reeves' study focused only on intestine and high zinc intake, our study included both small intestinal and hepatic M T changes in M T - W T mice in response to diets containing three different levels of zinc. In both studies, intestinal M T concentrations declined by day 21, and Reeves showed a continued decline to near-control (50 mg Zn/kg diet) values by day 42. It is therefore possible that the small intestinal M T concentration in our MT-WT mice would have continued to decline if treatment duration was prolonged. Currently, no other studies have shown the adaptation responses of hepatic M T to zinc intake. Although our results found that hepatic M T concentrations remained the same regardless of zinc intake after 21 days of treatment, it is not known whether hepatic M T in low-zinc M T - W T mice would have further decreased if the treatment duration was prolonged. The adaptation response of both hepatic and small intestinal M T to zinc intake regardless of zinc intake observed in this study suggests that: 1) at high-zinc intake, M T was induced as an initial defense mechanism, but prolonged high-zinc intake might induce other defensive mechanisms, such as changes in the rate of absorption and excretion through urine, intestinal secretion, and bile secretion; 2) at low-zinc intake, M T was first induced due to the disturbed homeostasis; however, long-term low-zinc intake caused M T to degrade and donate its zinc to compensate for the low-zinc intake, which led to the decrease in M T concentration (Dalton et al., 1996); and 3) the adaptation response of M T occurred between 7 and 21 days 73 after treatment for both organs. However, the two organs might reach their respective homeostatic conditions at different time points. Zinquin is a useful tool in assessing the distribution of histochemically reactive zinc in tissues The probe used in this study is Zinquin (2-methyl-8-/?-toluenesulfonamido-quinoline), a membrane-permeable zinc-specific fluorescent probe sensitive to nanomolar free zinc (Zalewski et al., 1993; Truong-Tran et al., 2003). Zinquin is a derivative of TSQ (6-methoxy-8-p-toluenesulfonamido-quinoline), and has been widely used as a zinc-specific fluorescent for assessing histochemical reactive zinc (Nasir et al.1999; Zalewski et al., 1994). Zinquin reacts with free zinc ions and loosely-bound zinc to give strong Zinquin-dependent fluorescent signal (Ranaldi et al., 2002). The fluorescent Zinquin-zinc complex can be examined using digital image analysis or spectrofluorimetry (Zalewski et al., 1993; Zalewski et al, 1994). Zinquin is a good tool for examining histochemically reactive zinc because: 1) it is zinc specific. An X-ray structure of Zinquin-Zn 2 + complex reveals a tetrahedral coordination of the metal center. The 2-methyl substituents and the unusual small bite angle (N-Zn-N) are responsible for Zinquin's high selectivity (Nasir et al., 1999); 2) it detects loosely-bound zinc besides free zinc ions, but not the tightly-bound zinc (Zalewski et al., 1994); and 3) it is relatively sensitive in detecting small changes of zinc (Zalewski et al., 1993). Furthermore, the activity of Zinquin can retain for several hours, and our preliminary results found that Zinquin-stained tissue slices maintained their fluorescence and intensity for as long as 24 hours. Zinquin is therefore widely used to examine the intracellular and tissue distribution and abundance of histochemically reactive zinc in a variety of tissues, including 74 the brain (Snitsarev et al., 2001) and pancreas (Zalewski et al , 1994). Evidently, Zinquin is a useful tool in assessing the relative abundance and distribution of histochemically reactive zinc. However, Zinquin is not a ratiometric fluorescent probe. Therefore, the overall brightness of the image and the relative brightness between the images are used to estimate the abundance of histochemically reactive zinc. The abundance of histochemically reactive zinc in M T - W T mice varies in response to zinc intake and treatment duration in small intestine but not in the liver The overall and relative brightness of the images are used to estimate the relative abundance of histochemically reactive zinc. To minimize the subjectivity in ranking the overall and relative brightness of the images, we invited eight individuals who had no knowledge of the experiment to rank the brightness of the images. To further ensure objectivity, these eight people performed the ranking separately. At low-zinc intake, the abundance of small intestinal histochemically reactive zinc in low-zinc M T - W T mice was generally lower than that in adequate-zinc M T - W T mice, except on day 21. On day 21, the abundance of small intestinal histochemically reactive zinc in low-zinc MT-WT mice was similar to that in adequate-zinc MT-WT mice. At high-zinc intake, the abundance of small intestinal histochemically reactive zinc was generally higher in high-zinc MT-WT mice than that in adequate-zinc MT-WT mice. These observations are supported by Paski et al's (2003) observations. In their study, feeding a low-zinc diet (3 mg Zn/kg diet) for 2 and 6 weeks reduces the abundance of small intestinal histochemically reactive zinc in rats, while zinc supplementation (155 mg Zn/kg diet) for the same duration increases the abundance of small intestinal histochemically reactive zinc. Small intestine is a 75 major zinc absorption site and plays an important role in zinc homeostasis, but the mechanisms involved in zinc absorption in small intestine are far from clear. In general, zinc is absorbed through a carrier-mediated process at low-zinc intake and a passive non-carrier mediated process (i.e. passive diffusion) at high-zinc intake (Davis, 1980; Groff and Gropper, 1999). Total zinc concentration in small intestine reflects dietary zinc intake, especially at high-zinc intakes. For example, small intestinal zinc concentration in rats fed excess-zinc diet (1000 mg Zn/kg diet) is 136% higher than those fed normal-zinc diet (100 mg Zn/kg diet) and 173% higher than those fed low-zinc diet (Tran et al., 1999). In mice, zinc supplementation (150 mg Zn/kg diet) also increases small intestinal zinc concentration by 35% compared to low-zinc intake (10 mg Zn/kg diet; Tran et al., 1998). Our observations show an association between the dietary zinc intake and the abundance of histochemically reactive zinc in small intestine in MT-WT mice. In contrast, abundance of histochemically reactive zinc in the liver in MT-WT mice was not changed in response to dietary zinc intake, except on day 7 in high-zinc MT-WT mice. In MT-WT mice after 7 days of high-zinc intake, the abundance of hepatic histochemically reactive zinc was higher than that in their corresponding adequate-zinc M T -WT mice. The lack of influence of dietary zinc intake on the abundance of hepatic histochemically reactive zinc may reflect the fact that hepatic zinc concentration is generally unaffected by dietary zinc intakes. For example, unlike other tissues such as bone and hair, zinc concentration in the liver is rapidly normalized at low-zinc intake (Hambidge et a l , 1986) and remains unchanged even during severe zinc deficiency (Taylor & Bray, 1991; Xu et a l , 1994). At high-zinc intake, hepatic zinc concentration also remains unaffected (Hambidge et al , 1986). Hepatic zinc concentration in rats fed 10 to 1000 mg Zn/kg diet 76 remains relatively constant after 7 days of treatment (Tran et al., 1999). In M T - T G mice, 6 5 Z n uptake in the liver is also unaffected by oral gavage at 10, 50, or 100 ug of zinc as ZnSO*4 (Coyle et al., 2000). Therefore, like total hepatic zinc concentration, the abundance of hepatic histochemically reactive zinc in MT-WT mice appears to be unaffected by dietary zinc intakes at intake levels tested. Metallothionein-bound zinc appears to be part of the histochemically reactive zinc pool in small intestine, but this relationship is less clear in the liver In small intestine, the abundance of histochemically reactive zinc responded to zinc treatment and treatment duration in a similar pattern as M T concentration did. Regardless of treatment duration, the abundance of small intestinal histochemically reactive zinc was generally lower in low-zinc MT-WT mice than that in adequate-zinc MT-WT mice. The lower abundance of small intestinal histochemically reactive zinc observed in low-zinc M T -WT mice coincided with the lower small intestinal M T concentration in low-zinc MT-WT mice compared to adequate MT-WT mice. The abundance of small intestinal histochemically reactive zinc was generally higher in high-zinc MT-WT mice than that in adequate-zinc MT-WT mice. Again, the higher abundance of small intestinal histochemically reactive zinc observed in high-zinc MT-WT mice coincided with the higher M T concentration in high-zinc MT-WT mice compared to adequate-zinc MT-WT mice. Similarly, the increase in the abundance of small intestinal histochemically reactive zinc over treatment duration coincided with the increase in small intestinal M T concentration over treatment duration in all three zinc treatment groups, especially in high-zinc MT-WT mice. Moreover, when small intestinal M T concentration decreased from day 7 to day 21 of 77 treatment, the abundance of small intestinal histochemically reactive zinc also decreased during the same period in all three zinc treatment groups. Using immunohistochemical technique, M T has been shown to predominantly present in the same anatomical area, where majority of histochemically reactive zinc accumulates (Tran et al., 1999; Szezurek et al., 2001). This apparent association between the abundance of small intestinal histochemically reactive zinc and small intestinal M T concentration suggests that MT-bound zinc contributes to the histochemically reactive zinc pool in small intestinal in M T - W T mice. If MT-bound zinc contributes to the abundance of histochemically reactive zinc in small intestine, a lack of M T should result in a lower abundance of histochemically reactive zinc. Indeed, the abundance of small intestinal histochemically reactive zinc in M T - K O mice was generally lower than that in MT-WT mice prior to zinc treatment and at all the levels of zinc intake tested. The steady level of zinc transporters mRNA is not affected by zinc intake in MT-WT mice Currently, the effect of dietary zinc intake on the expression of transporters involved in zinc influx (e.g. Nramp2) and efflux (e.g. ZnTs) is much understudied and the findings are unclear. In general, the expressions of these zinc transporters do not appear to be responsive to dietary zinc intakes. For example, there is a lack of association between zinc intakes and Nramp transporters (Tandy et a l , 2000; Sacher et al., 2001). In mice, the abundance of ZIP4 mRNA in small intestine increases during zinc deficiency and decreases in response to zinc supplement, but the abundance of ZIPl, 2, and 3 mRNA levels are unaffected by dietary zinc intake (Dufner-Beattie et al., 2003; Wang et al., 2004). In rats, small intestinal ZnT2 mRNA 78 level, but not ZnT-1 and ZnT-4 mRNA levels, generally reflects dietary zinc intakes (Luizzi et al., 2001). However, in the liver, none of these transporters examined responds to zinc intake (Liuzzi et al., 2001). The abundance of hepatic ZnT-1 mRNA level was unaffected by inadequate-zinc intake or zinc supplementation (McMahon and Cousins, 1998). In this study, the data also showed a lack of clear influence of dietary zinc intake on the abundance of zinc transporters assessed. This lack of apparent dietary zinc effect on the expression of zinc transporters may suggest that the expressions of these zinc transporters are under complex regulations, including tissue-specific and transporter-specific regulations. Moreover, the possibility of post-transcriptional modifications for these zinc transporters should also be taken into consideration. Systemic studies are thus needed to elucidate the influence of dietary zinc intake on the expression of these zinc transporters. M T - K O mice might decrease the abundance of zinc exporter m R N A and increase the abundance of importer mRNA to compensate for the absence of M T Although the zinc transporters we examined in MT-WT and M T - K O mice did not reflect dietary zinc intake in any specific fashion, differences between MT-WT and M T - K O mice were observed in ZnT-2, ZnT-4, and Nramp2 mRNA levels. No difference in ZnT-1 mRNA level, however, was observed between MT-WT and M T - K O mice. This observation is consistent with other studies, in which they found no direct association between M T and ZnT-1 (Davis et a l , 1998; Krebs, 2000). The abundance of hepatic and intestinal ZnT-1 mRNA was found to be similar in M T - T G and M T - K O mice (Davis et a l , 1998). Our finding therefore further supports a lack of association between M T and ZnT-1 mRNA level. 79 Besides ZnT-1, no other zinc transporters have been studied in their associations with M T thus far. Therefore, our findings first demonstrate a possible association between ZnT-2, ZnT-4, Nramp2 and MT. In M T - K O mice, significantly lower ZnT-2 and ZnT-4 (exporters) mRNA levels and significantly higher Nramp2 (importer) mRNA level were found compared to those in MT-WT mice. The findings are consistent with MT's function as a zinc absorber and reservoir. Various in vitro and in vivo studies have shown that as zinc concentration in growing medium or zinc intake increases, an elevation in M T accompanied by an increase in intracellular zinc or total tissue zinc are observed. M T facilitates zinc uptake and retention, especially under zinc-limiting conditions (Suhy et al., 1999; Philcox et al., 2000). In Davis et al.'s (1998) study, M T - K O mice has significantly higher intestinal zinc compared to MT-WT after zinc treatment. Therefore, based on MT's function as a zinc absorber and reservoir and the findings from previous studies, it is possible that in our study, Nramp2 mRNA was upregulated to import zinc in M T - K O mice to accommodate for the absence of MT. Meanwhile, ZnT-2 and ZnT-4 mRNA were down-regulated in M T - K O mice to enhance cellular zinc retention. The lowered ZnT-2 and ZnT-4 mRNA levels might have also contributed to the zinc toxicity observed in M T - K O mice at high-zinc intake. Without having M T to provide a cytoprotective role, M T - K O mice's sensitivity for high-zinc intake increases (Suhy et al., 1999); however, the lowered ZnT-2 and ZnT-4 mRNA level impeded the cells from exporting excessive zinc. The absence of M T , the upregulated importer, and the down-regulated zinc exporters probably all contributed to zinc toxicity in M T - K O mice at high-zinc intake. However, studies measuring the protein levels of the transporters are needed to further provide evidence for the association between zinc transporters and MT. 80 In summary, we have observed that in MT-WT mice, both M T concentration and the abundance of histochemically reactive zinc in small intestine reflected dietary zinc intake. The close association between M T and histochemically reactive zinc in small intestine and the lower abundance of histochemically reactive zinc in M T - K O mice suggest that a portion of MT-bound zinc is part of histochemically reactive zinc pool. The expressions of ZnT-1, ZnT-2, ZnT-4, and Nramp2, on the other hand neither reflected dietary zinc intake nor associated with the abundance of histochemically reactive zinc. Overall, the results obtained from this study suggest that M T , not zinc transporters examined, is involved in the homeostatic regulation of histochemically reactive zinc in mice. 81 Table H - l . Effect of dietary zinc intake on body weight gain and feed intake in M T - W T and M T - K O mice1. Treatment Body Weight Gain (g/mouse/day) Feed Intake (g/mouse/day) Duration Zn2 Zn30 Znl50 Zn2 Zn30 Znl50 M T - W T 2d nd nd nd 1.19 ± 0.21A (7) 1.45±0.25A (6) 1.69±0.16A (6) 4d 0.33 ± 0.05 (7) 0.35 ± 0.05 (6) 0.29 ± 0.07 (5) 2.12±0.16B (7) 2.48±0.14B (6) 2.27±0.11 B (5) 7d 0.21 ±0.10 (7) 0.28 ±0.05 (6) 0.30 ± 0.04 (5) 3.03 ± 0.09c (7) 3.11 ± 0.12c (6) 3.17±0.11 c (5) 21d 0.33 ± 0.02 (7) 0.40 ± 0.03 (5) 0.33 ± 0.05 (6) 3.21 ±0.08° (7) 3.86 ± 0.07° (5) 3.70 ± 0.11° (6) M T - K O 4d 0.14±0.03 a b (6) 0.24 ± 0.08a (5) 0.06 ± 0.30b* (5) 1.46 ±0.37 (6) 1.01 ± 0.43* (5) 1.02 ±0.51* (5) 1 Values are mean ± SEM. Number of replications is indicated in bracket, nd = not determined. Zn2: low-zinc; Zn30: adequate-zinc; Znl50: high-zinc. MT-WT: metallothionein wild type mice; MT-KO: metallothionein knockout mice. Upper case letters indicate significant differences in MT-WT mice in the same treatment group across time points (p < 0.05). Lower case letters indicate significant differences among dietary zinc treatment groups in MT-KO (p < 0.05). Lack of letters indicates no significant differences. Means sharing the same upper or lower case letter are not significantly different. The asterisk (*) indicates significant differences between MT-KO mice and the corresponding MT-WT control mice after 4 days of dietary zinc treatment (p < 0.05). 70 60 50 "o c o •Q 40 TJ S> 30 c N 20 10 EB Chow • Zn2 • Zn30 HZn150 BC C D E MT-WT Od EF CD T FG CDE i 1 II * c be ab 1 MT-WT MT-WT MT-WT MT-KO MT-KO 4d 7d 21 d Od 4d Figure II-l. Effect of dietary zinc intake and treatment duration on bone zinc concentration. V a l u e s are m e a n ± S E M ( n = 5 - 7). C h o w : l abora tory rodent c h o w ; Z n 2 : l o w - z i n c ; Z n 3 0 : adequate-z inc; Z n l 5 0 : h i g h - z i n c . M T - W T : m e t a l l o t h i o n e i n w i l d type m i c e ; M T - K O : m e t a l l o t h i o n e i n k n o c k o u t mice . U p p e r case letters indicate s igni f icant d i f ferences i n M T - W T m i c e a m o n g different z i n c treatments a n d treatment durat ions (p < 0.05). L o w e r case letters indicate s igni f icant di f ferences a m o n g z i n c treatment g r o u p s i n M T - K O (p < 0.05). M e a n s shar ing the same u p p e r or l o w e r case letter are not s ign i f i cant ly different. T h e n u m b e r s i g n (#) indicates s igni f icant d i f ferences b e t w e e n M T - K O m i c e a n d the c o r r e s p o n d i n g M T - W T contro l m i c e o n d a y 0. T h e asterisk (*) indicates s igni f icant d i f ferences be tween M T - K O m i c e a n d the c o r r e s p o n d i n g M T - W T c o n t r o l m i c e after 4 days o f t r e a t m e n t ( p < 0 . 0 5 ) . 83 Figure H-2. Effect of dietary zinc intake and treatment duration on metallothionein level in the liver. Values are mean ± SEM (n = 5 - 7). Chow: laboratory rodent chow; Zn2: low-zinc; Zn30: adequate-zinc; Znl50: high-zinc. MT-WT: metallothionein wild type mice; M T - K O : metallothionein knockout mice. Upper case letters indicate significant differences in M T - W T mice among different zinc treatments and treatment durations (p < 0.05). Means sharing the same upper case letter are not significantly different. 84 60 50 | 4 0 a> o>30 20 10 0 H Chowl • Zn2 E!Zn30 K3Zn150 FG MT-WT 2d B EF F G MT-WT 4d MT-WT 7d E F G MT-WT 21d Figure n-3. Effect of dietary zinc intake and treatment duration on metallothionein level in small intestine. Values are mean ± SEM (n = 5 - 7). Chow: laboratory rodent chow; Zn2: low-zinc; Zn30: adequate-zinc; Znl50: high-zinc. MT-WT: metallothionein wild type mice; M T - K O : metallothionein knockout mice. Upper case letters indicate significant differences in M T - W T mice among different zinc treatments and treatment durations (p < 0.05). Means sharing the same upper case letter are not significantly different. 85 86 Figure II-4. Zinquin-dependent fluorescence of the liver in MT-WT mice. A: basal level in M T - W T mice; B, C, and D: MT-WT mice after 2 days of low-, adequate-, and high-zinc intakes, respectively; E , F, and G: MT-WT mice after 4 days of low-, adequate-, and high-zinc intake, respectively; H, I, and J: M T - W T mice after 7 days of low-, adequate-, and high-zinc intake, respectively; K, L , and M : M T - W T mice after 21 days of low-, adequate-, and high-zinc intake, respectively; AA: basal level in M T - K O mice; BB, C C , and DD: M T - K O mice after 4 days of low-, adequate-, and high-zinc intake, respectively. The images were photographed at 100 x magnification using the same exposure time and aperture. 87 88 Figure II-5. Zinquin-dependent fluorescence of small intestine in MT-WT mice. A: basal level in M T - W T mice; B, C, and D: M T - W T mice after 2 days of low-, adequate-, and high-zinc intakes, respectively; E , F, and G: M T - W T mice after 4 days of low-, adequate-, and high-zinc intake, respectively; H, I, and J: MT-WT mice after 7 days of low-, adequate-, and high-zinc intake, respectively; K, L, and M : M T - W T mice after 21 days of low-, adequate-, and high-zinc intake, respectively; AA: basal level in M T - K O mice; BB, CC, and DD: M T -K O mice after 4 days of low-, adequate-, and high-zinc intake, respectively. The images were photographed at 200 x magnification using the same exposure time and aperture. 89 ZnT-1/p-actin ratio (arbitrary units) O O - * o i > b o k i c n t o so o Figure II-6. R T - P C R analysis on the effect of dietary zinc intake and treatment duration on ZnT-1 m R N A level in the liver. Total RNAs were isolated from the liver and were reverse transcribed as described in Material and Methods. PCR products were subjected to agarose (1.5%) gel electrophoresis. (A) Ethidium bromide-stained agarose gel showing representative hepatic ZnT-1 mRNA levels in MT-WT mice. Lane 1: lOObp D N A ladder; Lane 2: negative control (no cDNA); Lane 3: negative control (no primer); Lane 4: basal level; Lanes 5, 6, and 7: 2 days after low-, adequate-, and high-zinc intake, respectively; Lanes 8,9, and 10:4 days after low-, adequate-, and high-zinc intake, respectively; Lanes 11, 12, and 13:7 days after low-, adequate-, and high-zinc intake, respectively; Lanes 14,15, and 16: 21 days after low-, adequate, and high-zinc intake, respectively. (B) Ethidium bromide-stained agarose gel showing representative hepatic ZnT-1 mRNA levels in M T - K O mice. Lane 1: lOObp D N A ladder; Lane 2: negative control (no cDNA); Lane 3: negative control (no primer); Lane 4: basal level; Lanes 5, 6, and 7: 4 days after low-, adequate-, and high-zinc intake, respectively. (C) Ethidium bromide-stained agarose gel showing representative hepatic (3-actin mRNA levels in MT-WT mice. Lane 1: lOObp DNA ladder; Lane 2: negative control (no cDNA); Lane 3: negative control (no primer); Lane 4: basal level; Lanes 5, 6, and 7: 2 days after low-, adequate-, and high-zinc intake, respectively; Lanes 8,9, and 10: 4 days after low-, adequate-, and high-zinc intake, respectively; Lanes 11, 12, and 13: 7 days after low-, adequate-, and high-zinc intake, respectively; Lanes 14, 15, and 16: 21 days after low-, adequate-, and high-zinc intake, respectively. (D) Ethidium bromide-stained agarose gel showing representative hepatic P-actin mRNA levels in M T - K O mice. Lane 1: lOObp DNA ladder; Lane 2: negative control (no cDNA); Lane 3: negative control (no primer); Lane 4: basal level; Lanes 5, 6, and 7: 4 days after low-, adequate-, and high-zinc intake, respectively. (E) Relative hepatic ZnT-1 mRNA level. Values are mean ± SEM, n = 4 mice. Chow: laboratory rodent chow; Zn2: low- zinc; Zn30: adequate-zinc; Znl50: high-zinc. MT-WT: metallothionein wild type mice; M T - K O : metallothionein knockout mice. Upper case letters indicate significant differences in MT-WT mice among at different zinc treatments and treatment durations (p < 0.05). Lower case letters indicate significant differences among zinc treatment groups in M T - K O mice (p < 0.05). Means sharing the same upper or lower case letter are not significantly different. 91 Figure H -7 . R T - P C R analysis on the effect of dietary zinc intake and treatment duration on ZnT-1 mRNA level in small intestine. Total RNAs were isolated from small intestine and were reverse transcribed as described in Material and Methods. PCR products were subjected to agarose (1.5%) gel electrophoresis. (A) Ethidium bromide-stained agarose gel showing representative small intestinal ZnT-1 mRNA levels in M T - W T mice. Lane 1: 100 bp D N A ladder; Lane 2: negative control (no cDNA); Lane 3: negative control (no primer); Lane 4: basal level; Lanes 5, 6, and 7: 2 days after low-, adequate, and high-zinc intake, respectively; Lanes 8, 9, and 10: 4 days after low-, adequate-, and high-zinc intake, respectively; Lanes 11, 12, and 13: 7 days after low-, adequate-, and high-zinc intake, respectively; Lanes 14, 15, and 16: 21 days after low-, adequate-, and high-zinc intake, respectively. (B) Ethidium bromide-stained agarose gel showing representative small intestinal ZnT-1 mRNA levels in M T - K O mice. Lane 1: lOObp D N A ladder; Lane 2: negative control (no cDNA); Lane 3: negative control (no primer); Lane 4: basal level; Lanes 5, 6, and 7: 4 days after low-, adequate-, and high-zinc intake, respectively. (C) Ethidium bromide-stained agarose gel showing representative small intestinal P-actin mRNA levels in M T - W T mice. Lane 1: lOObp D N A ladder; Lane 2: negative control (no cDNA); Lane 3: negative control (no primer); Lane 4: basal level; Lanes 5, 6, and 7: 2 days after low-, adequate-, and high-zinc intake, respectively; Lanes 8,9, and 10: 4 days after low-, adequate-, and high-zinc intake, respectively; Lanes 11, 12, and 13: 7 days after low-, adequate-, and high-zinc intake, respectively; Lanes 14, 15, and 16: 21 days after low-, adequate, and high-zinc intake, respectively. (D) Ethidium bromide-stained agarose gel showing representative small intestinal p-actin mRNA levels in M T - K O mice. Lane 1: lOObp D N A ladder; Lane 2: negative control (no cDNA); Lane 3: negative control (no primer); Lane 4: basal level; Lanes 5, 6, and 7: 4 days after low-, adequate-, and high-zinc intake, respectively. (E) Relative small intestinal ZnT-1 mRNA level. Values are mean ± SEM, n = 4 mice. Chow: laboratory rodent chow; Zn2: low zinc; Zn30: normal zinc; Znl50: high zinc. MT-WT: metallothionein wild type mice; MT-KO: metallothionein knockout mice. Lack of letter indicates no significant differences. Lower case letters indicate significant differences among zinc treatment groups in M T - K O mice (p < 0.05). Means sharing the same lower or upper case letter are not significantly different. The number sign (#) indicates significant differences between M T - K O mice and the corresponding MT-WT control mice on day 0 (p < 0.05). 93 B 500 bp 400 bp 2d 4d 7d 21d — mm i g 1 2 3 4 5 6 7 3 9 10 11 12 13 14 15 16 MT-KO 500 bp 400 bp 1.6 o 1 f 1-2 c c I i g l 0 . 8 c N 0.4 B Chow • 2n2 • Zn30 S2n150 MT-WT 0d MT-WT 2d l l 1 MT-WT 4d i lab MT-WT 7d MT-WT 21 d MT-KO Od MT-KO 4d 94 Figure H-8. R T - P C R analysis on the effect of dietary zinc intake and treatment duration on ZnT-2 mRNA level in the liver. Total RNAs were isolated from the liver and were reverse transcribed as described in Material and Methods. PCR products were subjected to agarose (1.5%) gel electrophoresis. (A) Ethidium bromide-stained agarose gel showing representative hepatic ZnT-2 mRNA levels in M T - W T mice. Lane 1: 100 bp D N A ladder; Lane 2: negative control (no cDNA); Lane 3: negative control (no primer); Lane 4: basal level; Lanes 5, 6, and 7: 2 days after low-, adequate-, and high-zinc intake, respectively; Lanes 8, 9, and 10: 4 days after low-, adequate-, and high-zinc intake, respectively; Lanes 11, 12, and 13: 7 days after low-, adequate-, and high-zinc intake, respectively; Lanes 14,15, and 16: 21 days after low-, adequate-, and high-zinc intake, respectively. (B) Ethidium bromide-stained agarose gel showing representative hepatic ZnT-2 mRNA levels in M T - K O mice. Lane 1: lOObp DNA ladder; Lane 2: negative control (no cDNA); Lane 3: negative control (no primer); Lane 4: basal level; Lanes 5, 6, and 7: 4 days after low-, adequate-, and high-zinc intake, respectively. (C) Relative hepatic ZnT-2 mRNA level. Values are mean ± SEM, n = 4 mice. Chow: laboratory rodent chow; Zn2: low-zinc; Zn30: adequate-zinc; Znl50: high-zinc. MT-WT: metallothionein wild type mice; M T - K O : metallothionein knockout mice. Lack of letters indicates no significant differences. Lower case letters indicate significant differences among zinc treatment groups in M T - K O mice (p < 0.05). Means sharing the same lower or upper case letter are not significantly different. 95 Figure U-9. R T - P C R analysis on the effect of dietary zinc intake and treatment duration on ZnT-2 mRNA level in small intestine. Total RNAs were isolated from small intestine and were reverse transcribed as described in Material and Methods. PCR products were subjected to agarose (1.5%) gel electrophoresis. (A) Ethidium bromide-stained agarose gel showing representative small intestinal ZnT-2 mRNA levels in M T - W T mice. Lane 1: 100 bp D N A ladder; Lane 2: negative control (no cDNA); Lane 3: negative control (no primer); Lane 4: basal level; Lanes 5, 6, and 7: 2 days after low-, adequate-, and high-zinc intake, respectively; Lanes 8, 9, and 10: 4 days after low-, adequate-, and high-zinc intake, respectively; Lanes 11, 12, and 13: 7 days after low-, adequate-, and high-zinc intake, respectively; Lanes 14, 15, and 16: 21 days after low-, adequate-, and high-zinc intake, respectively. (B) Ethidium bromide-stained agarose gel showing representative small intestinal ZnT-2 mRNA levels in M T - K O mice. Lane 1: basal level; Lanes 2, 3, and 4: 4 days after low-, adequate-, and high-zinc intake, respectively; Lane 5: negative control (no cDNA); Lane 6: negative control (no primer); Lane 7. lOObp D N A ladder. (C) Relative small intestinal ZnT-2 mRNA level. Values are mean ± SEM, n = 4 mice. Chow: laboratory rodent chow; Zn2: low-zinc; Zn30: adequate-zinc; Znl50: high-zinc. Upper case letters indicate significant differences in MT-WT mice among different zinc treatments and treatment durations (p < 0.05). Lower case letters indicate significant differences among zinc treatment groups in M T - K O mice (p < 0.05). Lack of lower case letters indicates no significant difference in M T - K O mice. Means sharing the same lower or upper case letter are not significantly different. The asterisk (*) indicates significant differences between M T - K O mice and the corresponding MT-WT control mice after 4 days of dietary zinc treatment (p < 0.05). 97 B 600 bp 500 bp' 2d 4d 7d 21 d MT-KO 1 2 3 4 5 6 7 ! ! 9 10 11 12 13 14 15 16 600 bp 500 bp—* 1 1 2 3 4 5 6 7 1.6 33 2 c o ro • CO. I— c N * 1 c 3 e? _ .2 H .8 0.4 fflChow • Zh2 • Zn30 HZn150 FGH HI i AB -r ABC ABCD BCDE CDEF DEF EFG I 1 M T - W T M T - W T Od 2d M T - W T 7d M T - W T M T - K O M T - K O 21 d 0d 4d 98 Figure 11-10. R T - P C R analysis on the effect of dietary zinc intake and treatment duration on ZnT-4 mRNA level in the liver. Total RNAs were isolated from the liver and were reverse transcribed as described in Material and Methods. PCR products were subjected to agarose (1.5%) gel electrophoresis. (A) Ethidium bromide-stained agarose gel showing representative hepatic ZnT-4 mRNA levels in MT-WT mice. Lane 1: 100 bp D N A ladder; Lane 2: negative control (no cDNA); Lane 3: negative control (no primer); Lane 4: basal level; Lanes 5, 6, and 7: 2 days after low-, adequate-, and high-zinc intake, respectively; Lanes 8, 9, and 10: 4 days after low-, adequate-, and high-zinc intake, respectively; Lanes 11, 12, and 13: 7 days after low-, adequate-, and high-zinc intake, respectively; Lanes 14,15, and 16: 21 days after low-, adequate-, and high-zinc intake, respectively. (B) Ethidium bromide-stained agarose gel showing representative hepatic ZnT-4 mRNA levels in M T - K O mice. Lane 1: lOObp DNA ladder; Lane 2: negative control (no cDNA); Lane 3: negative control (no primer); Lane 4: basal level; Lanes 5, 6, and 7: 4 days after low-, adequate-, and high-zinc intake, respectively. (C) Relative hepatic ZnT-4 mRNA level. Values are mean ± SEM, n — 4 mice. Chow: laboratory rodent chow; Zn2: low-zinc; Zn30: adequate-zinc; Znl50: high-zinc. Upper case letters indicate significant differences in MT-WT mice among different zinc treatments and treatment durations (p < 0.05). Lower case letters indicate significant differences among zinc treatment groups in M T - K O mice (p < 0.05). Lack of lower case letters indicates no significant differences in M T - K O mice. Means sharing the same lower or upper case letter are not significantly different. The asterisk (*) indicates significant differences between M T - K O mice and the corresponding M T - W T control mice after 4 days of treatment (p < 0.05). 99 B 600 bp 500 bp 2d 4d 7d 21d MT-KO 600 bp 500 bp • 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 1 2 3 4 5 6 7 1.6 o c I ° ro £ i « t I I— 0^.8 c N 0.4 BChow • Zn2 • Zn30 SZn150 h i 1 MT-WT 0d MT-WT 2d i i i 1 MT-WT 4d 1 MT-WT 21 d MT-KO 0d MT-KO 4d 100 Figure 11-11. RT-PCR analysis on the effect of dietary zinc intake and treatment duration on ZnT-4 mRNA level in small intestine. Total RNAs were isolated from small intestine and were reverse transcribed as described in Material and Methods. PCR products were subjected to agarose (1.5%) gel electrophoresis. (A) Ethidium bromide-stained agarose gel showing representative small intestinal ZnT-4 mRNA levels in MT-WT mice. Lane 1: 100 bp DNA ladder; Lane 2: negative control (no cDNA); Lane 3: negative control (no primer); Lane 4: basal level; Lanes 5, 6, and 7: 2 days after low-, adequate-, and high-zinc intake, respectively; Lanes 8, 9, and 10: 4 days after low-, adequate-, and high-zinc intake, respectively; Lanes 11, 12, and 13: 7 days after low-, adequate-, and high-zinc intake, respectively; Lanes 14, 15, and 16: 21 days after low-, adequate-, and high-zinc intake, respectively. (B) Ethidium bromide-stained agarose gel showing representative small intestinal ZnT-4 mRNA levels in M T - K O mice. Lane 1: lOObp D N A ladder; Lane 2: negative control (no cDNA); Lane 3: negative control (no primer); Lane 4: basal level; Lanes 5, 6, and 7: 4 days after low-, adequate-, and high-zinc intake, respectively. (C) Relative small intestinal ZnT-4 mRNA level. Values are mean ± SEM, n = 4 mice. Chow: laboratory rodent chow; Zn2: low-zinc; Zn30: adequate-zinc; Znl50: high-zinc. Upper case letters indicate significant differences in MT-WT mice in the same treatment group across time points (p < 0.05). Lower case letters indicate significant differences among zinc treatment groups in M T - K O mice (p < 0.05). Lack of lower case letters indicates no significant differences in M T - K O mice. Means sharing the same lower or upper case letter are not significantly different. The asterisk (*) indicates significant differences M T - K O mice and the corresponding MT-WT control mice after 4 days of treatment (p < 0.05). 101 B 700 bp _ 600 bp" 2d 4d 7d 21d 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 700 bp 600 bp O i 1.6 1.2 += c O 3 <? £• ca. 2 E "0 .8 n 0.4 BChow • Zn2 • Zn30 KZn150 1 i MT-WT Od MT-WT 2d MT-WT 4d MT-WT 21 d MT-KO Od 1 MT-KO 4d 102 Figure 11-12. R T - P C R analysis on the effect of dietary zinc intake and treatment duration on Nramp2 mRNA level in the liver. Total RNAs were isolated from the liver and were reverse transcribed as described in Material and Methods. PCR products were subjected to agarose (1.5%) gel electrophoresis. (A) Ethidium bromide-stained agarose gel showing representative hepatic Nramp2 mRNA levels in MT-WT mice. Lane 1: 100 bp D N A ladder; Lane 2: negative control (no cDNA); Lane 3: negative control (no primer); Lane 4: basal level; Lanes 5, 6, and 7: 2 days after low-, adequate-, and high-zinc intake, respectively; Lanes 8, 9, and 10: 4 days after low-, adequate-, and high-zinc intake, respectively; Lanes 11, 12, and 13: 7 days after low-, adequate-, and high-zinc intake, respectively; Lanes 14, 15, and 16: 21 days after low-, adequate-, and high-zinc intake, respectively. (B) Ethidium bromide-stained agarose gel showing representative hepatic Nramp2 mRNA levels in M T - K O mice. Lane 1: lOObp DNA ladder; Lane 2: negative control (no cDNA); Lane 3: negative control (no primer); Lane 4: basal level; Lanes 5, 6, and 7: 4 days after low-, adequate-, and high-zinc intake, respectively. (C) Relative hepatic Nramp2 mRNA level. Values are mean ± SEM, n = 4 mice. Chow: laboratory rodent chow; Zn2: low-zinc; Zn30: adequate-zinc; Znl50: high-zinc. Upper case letters indicate significant differences in MT-WT mice in the same treatment group across time points (p < 0.05). Lower case letters indicate significant differences among zinc treatment groups in M T - K O mice (p < 0.05). Means sharing the same lower or upper case letter are not significantly different. The asterisk (*) indicates significant differences between M T - K O mice and the corresponding MT-WT control mice after 4 days of treatment (p < 0.05). 103 B 700 bp 600 bp 2 d 4d 7d 21d 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 700 bp 600 bp 1.6 o S _ fit" o =» 7 £> ca s 2 i Q. J= E -§ 0.8 0.4 • Chow • Zn2 • Zn30 HZn150 M T - W T Od M T - W T 2d II HI i * M T - W T M T - W T M T - W T M T - K O M T - K O 4d 7d 21d Od 4d 104 Figure 11-13. R T - P C R analysis on the effect of dietary zinc intake and treatment duration on Nramp2 mRNA level in small intestine. Total RNAs were isolated from small intestine and were reverse transcribed as described in Material and Methods. PCR products were subjected to agarose (1.5%) gel electrophoresis. (A) Ethidium bromide-stained agarose gel showing representative small intestinal Nramp2 mRNA levels in MT-WT mice. Lane 1: 100 bp D N A ladder; Lane 2: negative control (no cDNA); Lane 3: negative control (no primer); Lane 4: basal level; Lanes 5, 6, and 7: 2 days after low-, adequate, and high-zinc intake, respectively; Lanes 8, 9, and 10: 4 days after low-, adequate-, and high-zinc intake, respectively; Lanes 11, 12, and 13: 7 days after low-, adequate-, and high-zinc intake, respectively; Lanes 14, 15, and 16: 21 days after low-, adequate-, and high-zinc intake, respectively. (B) Ethidium bromide-stained agarose gel showing representative small intestinal Nramp2 mRNA levels in M T - K O mice. Lane 1: basal level; Lanes 2, 3, and 4: 4 days after low-, adequate-, and high-zinc intake, respectively; Lane 5: negative control (no cDNA); Lane 6: negative control (no primer); Lane 7: lOObp D N A ladder. (C) Relative small intestinal Nramp2 mRNA level. Values are mean ± SEM, n = 4 mice. Chow: laboratory rodent chow; Zn2: low-zinc; Zn30: adequate-zinc; Znl50: high-zinc. Upper case letters indicate significant differences in M T - W T mice in the same treatment group across time points (p < 0.05). Lower case letters indicate significant differences among zinc treatment groups in M T - K O mice (p < 0.05). Means sharing the same lower or upper case letter are not significantly different. The asterisk (*) indicates significant differences between M T - K O mice and the corresponding M T - W T control mice after 4 days of treatment (p < 0.05). 105 C H A P T E R HI F U T U R E D I R E C T I O N S 1. Metallothionein is part of the histochemically reactive zinc It has been shown that LEPZ and MT-bound zinc represent two different, but inter-related pools of zinc, as a portion of the MT-bound zinc contributes to the histochemically reactive zinc pool (Paski et al., 2003). In this study, we observed an association between the abundance of histochemically reactive zinc and M T concentration in small intestine in M T -WT mice. Moreover, compared to MT-WT mice, the abundance of both hepatic and small intestinal histochemically reactive zinc were lower in M T - K O mice regardless of zinc intake. These observations affirm the notion that MT-bound zinc contributes to the histochemically reactive zinc pool. However, in order to firmly establish the relationship between M T and histochemically reactive zinc, it is important to conduct studies using metallothionein-transgenic (MT-TG) mice. Like M T - K O mice, M T - T G mice are generally in good health and are visually indistinguishable from their genetic control mice. M T - T G mice have higher basal M T concentrations than their genetic control mice. M T in M T - T G mice is also inducible by zinc (Iszard et a l , 1995). Therefore, comparing the changes in the abundance of histochemically reactive zinc and M T concentration in response to zinc treatment between M T - T G mice and their genetic control mice can provide a more completed understanding of the relationship between M T and histochemically reactive zinc. 106 2. MT -3 and MT -4 proteins might have played an important role in the regulation of zinc homeostasis in M T - K O mice In M T - K O mice, although genes encoding MT-1 and MT-2 are lacking, genes encoding MT-3 and MT-4 are still expressed. MT-3 and MT-4 are tissue specific, with high abundance of MT-3 found in the brain. Since M T - K O mice are generally healthy and have higher bone zinc concentrations at low- or adequate-zinc intake than MT-WT mice in our study, it is speculated that MT-3 and MT-4 might have played a role in maintaining zinc concentration by retaining more zinc to compensate for the absence of MT-1 and MT-2 in M T - K O mice. Studies investigating the role of MT-3 and MT-4 in M T - K O mice, such as brain zinc concentration, the abundance of histochemically reactive zinc in the brain, MT-3 and MT-4 concentrations, can provide better understandings in the homeostatic regulation of zinc in M T - K O mice. 3. M T - K O mice might have used zincosomes as a strategy to control zinc toxicity Zincosomes are intracellular vesicles that are believed to contain a high density of zinc. Our knowledge about zincosomes, such as their characteristics and functions, is extremely limited. It is suggested that intracellular zinc ions are stored in zincosomes. Fluorescent staining using Zinquin shows a high amount of labile zinc in zincosomes in C6 rat glioma cells (Haase and Beyersmann, 2002). In our study, clear bright fluorescent "spots" were observed in the liver of M T - K O mice at high-zinc intake. These bright "spots" indicated an accumulation of labile zinc or Zinquin-reactive zinc. Based on the high fluorescence intensity, it is possible that these bright "spots" are a collection of cells with high histochemically reactive zinc. It is unknown as to why these clusters of cells had higher 107 abundance of histochemically reactive zinc. Moreover, since these bright "spots" were only observed in M T - K O mice at high-zinc intake, it is possible that at high-zinc intake, M T - K O mice might have attempted to reduce free ionic zinc concentration by storing zinc in zincosomes, hence the presence of bright "spots." However, since high-zinc intake caused high mortality rate in M T - K O mice, zincosomes might not be efficiently effective in removing free zinc ions at high-zinc intake. Further studies are warranted to investigate the role of zincosomes in protecting M T - K O mice from potential zinc toxicity. 108 R E F E R E N C E S Ackland, M.L. & Mercer, J.F.B. (1992) The murine mutation, lethal milk, results in production of zinc-dependent milk. J. 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C , Saari, J.T. & Kang, Y.J. (2002) Metallothionein-independent zinc protein from alcoholic liver injury. Am. J. Path., 160: 2267-2274. 123 A P P E N D I X I Dietary Composition 124 Table AI-1. Dietary Composition Ingredients Dietary Composition Low-zinc Adequate-zinc High-zinc (g/kg diet) Cornstarch 389.82 380.49 340.49 Egg white solids 200.00 200.00 200.00 Dextrinized cornstarch 132.00 132.00 132.00 Sucrose 100.00 100.00 100.00 soybean oil 70.00 70.00 70.00 Fiber 50.00 50.00 50.00 Mineral premix1 35.00 35.00 35.00 Vitamin premix2 10.00 10.00 10.00 Zinc premix3 0.67 10.00 50.00 Biotin premix4 10.00 10.00 10.00 Choline bitartrate 2.50 2.50 2.50 1 The composition of the mineral premix is listed in Table AI-2. 2 The composition of the vitamin premix is listed in Table AI-3. 3 The composition of the zinc premix is listed in Table AI-4. 4 The composition of biotin premix is listed in Table AI-5. 125 Table AI-2. Composition of mineral premix without zinc M I N E R A L P R E M I X Minerals g/kg mix Sucrose 399.01 CaCC»3, anhydrous 83.56 CaHPO-4 376.40 Kcitrate, tripotassium 108.09 MgO 24.00 Fe citrate 6.06 Na meta-silicate.9H20 1.45 M n C 0 3 0.63 CuC03 0.30 CrK(S0 4) 2 .12H 20 0.28 H3BO3 81.5 mg NaF 63.5 mg N1CO3 31.8 mg LiCl 17.4 mg KIO3 10.0 mg Ammonium paramolybdate.4H20 7.95 mg Ammonium vanadate 6.60 mg 126 Table AI-3. Composition of vitamin premix VITAMIN PREMIX Vitamins g/kg mix Sucrose 974.66 Nicotinic acid 3.00 Ca Pantothenate 1.60 Pyridoxine-HCl 0.70 Thiamin-HCl 0.60 Riboflavin 0.60 Folic acid 0.20 Biotin 0.02 Vitamin B-12 (0.1% in mannitol) 2.50 Vitamine E (500IU/g) 15.00 Vitamin A (500,000IU7g) 0.80 Vitamin D-3 (400,000IU/g) 0.25 Vitamin K - l (phylloquinone) 0.08 127 Table AI-4. Composition of zinc premix Ingredients g/kg mix Z n C 0 3 5.75 Cornstarch 994.25 Table AI -5. Composition of biotin premix Ingredients g/kg mix Biotin 0.18 Cornstarch 999.82 128 A P P E N D I X II Metallothionein concentration in M T - K O mice 129 Table A l l . Metallothionein concentration in M T - K O mice 1 Liver Small Intestine Ug M T / g tissue M T - K O mice 0.6 0.1 l rTo confirm the absence of MT-1 and MT-2 proteins in M T - K O mice, livers and small intestines from two M T - K O mice were removed for determining M T concentration using 109Cd-hemoglobin affinity assay. M T - K O mice had free access to regular laboratory rodent chow, which contained -70 mg Zn/kg diet. 130 

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